Small molecules blocking histone reader domains

ABSTRACT

The present invention relates to small molecules interfering with the conformational space of the TONSL ARD occupied by the histone H4 tail. These small molecules targets the binding pocket of TONSL encompassing the H4 residues K12-R23 and act by preventing or disrupting the binding of the H4 tail K12-R23 with the TONSL ARD via direct competition or via allosteric disruption of the binding pocket. The present inventors have identified and solved the structure of a histone reader domain of TONSL termed the ARD (ankyrin repeat domain).

FIELD OF THE INVENTION

The present invention relates to novel inhibitors of the TONSL protein required for genome stability. TONSL functions as an obligate heterodimer with MMS22L in DNA repair of double strand breaks (DSBs) and stabilization of replicating DNA intermediates via error-free homologous recombination (HR).

The present inventors have solved the structure of the TONSL ARD in complex with its histone substrate, histone H4 tails, providing grounds for structure-based rational drug design of small molecules, peptides or polypeptides capable of preventing or disrupting the binding of the H4 tail with the TONSL ARD via direct competition or via allosteric disruption of the binding pocket.

BACKGROUND OF THE INVENTION

Cells are continuously exposed to diverse sources of DNA damage, and to face this challenge they have devised an array of DNA repair strategies. One of the key repair pathways for DNA double strand breaks, damaged replication forks and key for survival of proliferating cells is homologous recombination (HR).

Many cancer cells experience high load of replication stress, making them vulnerable to HR inhibition.

Cancer cells often also depend on this pathway to repair DNA damage generated by conventional chemotherapy; therefore, HR targeting drugs are clinically attractive due to synthetic lethality of affected cells.

Development of HR inhibitors is also of great clinical interest because PARP inhibitors kill HR defective cells. Therefore blocking two alternative pathways will deliver a much bigger impact than targeting either pathway alone. Additionally, some tumours cells are defective in other DNA repair pathways and therefore dependent on HR, making them sensitive to inhibition of HR repair.

TONSL (Tonsoku-like, NFKBIL2) is a crucial HR regulator that functions in a heterodimeric complex with MMS22L to promote repair of DSBs and damaged replication forks by HR. In HR, TONSL-MMS22L is proposed to regulate Rad51 loading onto ssDNA. Depletion of MMS22L or TONSL results in a pronounced decrease of cell proliferation and marked hypersensitivity to the topoisomerase I poison camptothecin (CPT), which is most likely caused by an inability to promote RAD51-mediated repair of broken replication forks. However, the molecular mechanisms regulating TONSL-MMS22L function in DNA repair are not known.

TONSL-MMS22L forms a complex with histones H3-H4, the histone chaperone ASF1 and MCM2, 4, 6, 7 that depend on the TONSL Ankyrin Repeat Domain (ARD), suggesting that the ARD could be important for TONSL function.

ARD is a common protein-protein interaction motif consisting of tandemly repeated modules of about 33 amino acids, occurring in a large number of functionally diverse proteins including transcriptional initiators, cell cycle regulators, cytoskeletal proteins, ion transporters and signal transducers.

Crystal structures of several ARD proteins are solved, including G9a/GLP ARD that binds specifically histone H3 methylated at lysine 9 (Collins et al., Nat Struct Mol Cell Biol 15:245, 2008). Yet the specificity of most ARDs remain unknown.

By sequence similarity, TONSL ARD is highly similar to the BARD1 ARD. BARD1 is a well-characterized HR factor, obligate partner of BRCA1, and is required for most cellular and tumour-suppressor functions of BRCA1 (Westermark et al., Mol Cell Biol 23:7926, 2003).

The crystal structure of BARD1 ARD is available (Fox et al., J Biol Chem 283:21179, 2008) but the binding specificity of the BARD1 ARD is unknown, and thus does not provide grounds for inhibitor design.

Therefore, it is desirable to identify target molecules bound by TONSL ARD and obtain crystals of these protein complexes in order to solve their structures at atomic resolution. This provides basis to determine whether the binding is important for TONSL-MMS22L function, thus making it an attractive drug target. Further, high-resolution structures of binding pockets with their substrates can enable the skilled addressee in designing small molecules interfering with the binding of TONSL ARD to its target molecule and subsequently verifying the effect in biological assays in a rapid and reproducible manner.

SUMMARY OF THE INVENTION

The present inventors have discovered a molecular mechanism for recruitment of TONSL and its partner MMS22L to post-replicative chromatin that opens an avenue to target the HR repair pathway in cancer treatment. This mechanism relies on recognition of histone H4 unmodified at K20 (H4K2Ome0) by the TONSL Ankyrin Repeat Domain (ARD) domain that functions as a novel histone reader domain.

The highlights of this the discovery work include

-   -   1. Structural and biochemical data identifying the TONSL ARD as         the first histone reader specific for H4 tails unmethylated at         K20 (H4K20me0). H4K20me0 is specific to newly synthesized         histones incorporated during DNA replication, marking         post-replicative chromatin until late G2/M when K20me1 is         established.     -   2. Functional and structural data revealing that TONSL via ARD         recognition of H4K20me0 binds soluble new histones in complex         with MCM2 and ASF1, proving a means to deliver TONSL-MMS22L to         replicating chromatin.     -   3. Functional data that TONSL binds nucleosomal histones in         post-replicative chromatin via ARD recognition of H4K20me0 and         demonstration that this is required for TONSL-MMS22L         accumulation at damaged replication forks and DNA lesions.     -   4. Functional data that TONLS ARD mutant protein titrates MMS22L         away from chromatin and is toxic to cells.     -   5. The TONSL ARD mutant proteins induce G2/M arrest,         replication-associated DNA damage (53BP1 foci) and reduce         viability in the presence and absence of camptothecin (CPT).     -   6. By removing TONSL from chromatin, the function of         TONSL-MMS22L complex in DNA repair is disabled.     -   7. Inhibitors of TONSL, e.g inhibitors of TONSL capable of         associating with the TONSL ARD.     -   7. Histone H4 peptides or functional homologues thereof could         act as inhibitors of TONSL

Taken together, this shows that DNA replication leaves a mark, in the form of deposited new histones unmodified at H4K20, which are recognized by the TONSL-MMS22L HR repair complex to differentiate pre- and post-replicative chromatin. Furthermore, this work provides a breakthrough in understanding how the post-replicative chromatin state is read by a key HR factor TONSL, which mediates loading of Rad51 at damaged replication forks and DSBs. Small molecule inhibitors targeting TONSL ARD are thus capable to disable efficient HR repair of DSBs and damaged replication forks.

The present invention thus relates to small molecules interfering with the conformational space of the TONSL ARD occupied by the histone H4 tail. These small molecules targets the binding pocket of TONSL encompassing the H4 residues K12-R23 and act by preventing or disrupting the binding of the H4 tail K12-R23 with the TONSL ARD via direct competition or via allosteric disruption of the binding pocket.

Therefore, in one aspect the present invention relates to small molecules that prevent or disrupt the binding of a substrate in the binding pocket of the BARD1 ARD (defined by its high structural similarity to the H4 tail K12-R23 binding pocket in TONSL ARD) via direct competition or via allosteric disruption of the binding pocket.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified and solved the structure of a histone reader domain of TONSL termed the ARD (ankyrin repeat domain). Importantly, the present inventors have solved the structure of the TONSL ARD in complex with its histone substrate, histone H4 tails, providing grounds for structure-based rational drug design of TONSL inhibitors. Given the requirement of TONSL for HR of DSBs and repair of damaged replication forks, TONSL inhibitors can be efficient in compromising HR and be used either alone or in combination with conventional chemotherapy in killing cancer cells. Thus, any of the TONSL inhibitors described herein may be useful in the treatment of cancer.

The crystal structure of the complex, recombinant TONSL ARD, cell lines expressing GFP-TONSL (wild type and histone H4 binding mutants) of the present invention are all instrumental to design and identify specific small molecule inhibitors that interfere with TONSL ARD recognition of H4K20me0.

Below the present inventors, also disclose the development of a pipeline of biochemical and cell-based assays useful for TONSL inhibitor development. These data and information are indispensable for the design of novel inhibitors suitable as anti-cancer therapeutics. The invention thus provides the means for designing in silico and subsequently synthesizing and testing in assays also provided by the invention, small molecule inhibitors that are capable of inhibiting the binding between TONSL and its histone substrate, thereby interfering with HR, in turn resulting in direct anti-tumour effects and/or increased efficacy of available cytostatic compounds.

The present inventors have identified the key determinant for binding of H4 tails to TONSL using interfacial mutants. Furthermore, the present inventors have demonstrated that TONSL ARD mutants that disrupt the H4 tail binding sites are no longer recruited to post-replicative chromatin, damaged replication forks, or DNA lesions (e.g., DNA double strand breaks). The TONSL ARD mutant proteins induce G2/M arrest, replication-associated DNA damage (53BP1 foci) and reduce viability in the presence and absence of camptothecin (CPT).

As described in more detail below, said TONSL ARD mutants may phenocopy the effect of inhibitors of TONSL, in particular the effect of inhibitors which inhibit binding of TONSL ARD to histone H4. Thus, the invention provides that such inhibitors are useful for inducing G2/M arrest, replication-associated DNA damage (53BP1 foci) and/or reduce cell viability in the presence and/or absence of camptothecin (CPT). This render the inhibitors useful in the treatment of cancer. The inhibitor may be any of the inhibitors described herein.

The present inventors also note that several TONSL mutants in the ARD domain (residues 512-692) have been identified in cancer tissues of multiple organs including lung, skin, stomach, large intestine, biliary tract, prostate, endometrium, ovary, pancreas, oesophagus, urinary tract and central nervous system (COSMIC, Catalogue of somatic mutations in cancer, http://grch37-cancer.sang,ac.uk/cosmic/search?q=TONSL). The term TONSL ARD refers to amino acids 512 to 692 of SEQ ID NO:16.

The present inventors have determined that the TONSL ARD is highly similar to the ARD of BARD1 for which a structure has been published, but since the binding specificity of BARD1 ARD is unknown, this again does not provide grounds for inhibitor design.

However, the details of TONSL ARD binding to H4 tails predict that inhibitors that disrupt TONSL binding to histones may also bind the BARD1 ARD and disrupt interaction with its substrate (e.g. histones). This would be desirable and probably increase drug efficacy as BARD1 and TONSL operate in separate steps within the same DNA repair pathway (HR). The present inventors also note that an established tumour suppressor mutation in BARD1 found in breast cancer targets a key residue predicted to be required for histone binding on the basis of the TONSL ARD-H4 structure.

In one aspect, the present invention relates to designing and developing drugs, small molecule inhibitors that would prevent recruitment of TONSL, a HR protein, to sites of DNA damage. Because TONSL together with its partner protein MMS22L is required for HR, interfering with TONSL recruitment to DNA lesion will impair HR repair and kill cancer cells or cells dependent on HR repair pathway either alone or in combination treatment with conventional chemotherapy.

The Crystal Structure

The present invention relates to the use of structure-based drug design methods to identify compounds that interfere with TONSL and TONSL-MMS22L complex recruitment to DNA lesions and DNA replication forks.

The invention discloses the crystal structure of the TONSL ARD in complex with its histone substrate, histone H4 tails. The TONSL ARD targets the H4 tail spanning residues Lys12 to Arg23, primarily through intermolecular hydrogen-bonding, electrostatic and van der Waals interactions (FIGS. 1-9).

The invention relates to the atomic details of the TONSL ARD binding pocket architecture and the intermolecular interactions with the H4 tail peptide that account for recognition specificity between the H4 tail (K4-R23) and residues lining the TONSL binding pocket.

In a preferred embodiment, the present invention relates to a crystal structure having the atomic coordinates available in the PDB Protein Databank under the PDB ID 53A4 as deposited 11 Apr. 2016, DOI: 10.2210/pdb5ja4/pdb or having a structure in which the atomic coordinates vary by less than 3Å in any direction from those set out in the PDB Protein Databank under the PDB ID 5JA4, DOI: 10.2210/pdb5ja4/pdb.

The variation of the atomic coordinates of the present invention should preferably be less than 3Å, such as less than 2,75 Å, such as less than 2,5 Å, such as less than 2,25 Å, such as less than 2,0 Å, such as less than 1,75 Å, such as less than 1,5 Å, such as less than 1,25 Å, such as less than 1,0 Å, such as less than 0,75 Å, such as less than 0,5 Å or such as less than 0,25 Å.

In one embodiment, the invention relates to at least part of the atomic co-ordinate data available in the PDB Protein Databank under the PDB ID 53A4, DOI: 10.2210/pdb5ja4/pdb or data derivable therefrom.

In one embodiment, the present invention relates to a crystal comprising at least part of the crystal structure of the TONSL ARD in complex with its histone substrate, histone H4 tails.

In another embodiment, the invention relates to the distances between the atomic co-ordinates in the crystal structure available in the PDB Protein Databank under the PDB ID 5JA4, DOI: 10.2210/pdb5ja4/pdb or any variation thereof that defines the binding surface of TONSL interacting with the histone H4 tail residues Lys12-Arg23 (FIGS. 1-9).

The histone H4(K12 to R23) peptide is positioned within a channel on the surface of the ankyrin repeat domains of TONSL. This surface constitutes an acidic patch and contains shallow binding pockets for the His18 and Lys20 side chains of H4. Further, the side chain of H4 R17 is aligned by being sandwiched between the side chains of Tyr572 and Cys608. Hence, any inhibitor must target the two shallow pockets and be compatible with the electrostatic nature of the binding surface.

Methods of Forming a Crystal Structure

In one embodiment, the invention relates to a three dimensional crystal of a complex between

-   -   a polypeptide comprising:         -   TONSL ARD consisting of amino acids 512 to 692 of SEQ ID             NO:16 or a functional homologue thereof sharing at least 90%             sequence identity therewith; and         -   optionally MCM2 HBD consisting of amino acids 61 to 130 of             SEQ ID NO:24 or a functional homologue thereof sharing at             least 90% sequence identity therewith;     -   Histone H4 of SEQ ID NO:23 or a functional homologue thereof         sharing at least 90% sequence identity therewith; and     -   optionally Histone H3 or a fragment thereof, e.g. histone H3.3         Δ56 consisting of amino acids 57-135 of SEQ ID NO:25 or a         functional homologue thereof sharing at least 90% sequence         identity therewith.

In particular, the polypeptide may comprise said TONSL ARD or a functional homologue thereof linked to said MCM2 HBD or a functional homologue thereof by way of a peptide linker. Such a peptide linker typically consists of in the range 4 to 20 amino acids, e.g. 4 to 12 amino acids. Said amino acids may be Gly, and thus the linker may consist of in the range of 4 to 20 Gly residues, such as in the range of 4 to 12 Gly-residues.

In general, the complex comprises one polypeptide comprising TONSL ARD, whereas histone H4 and optionally histone H3 typically are present as separate peptides. Preferably, the polypeptide comprising TONSL ARD also comprise MCM2 HBD, wherein TONSL ARD may be linked to MCM2 HBD by way of a linker, e.g. a peptide linker. Said fragment of Histone H3 may be a fragment of histone H3.3, e.g. amino acids 57-135 of SEQ ID NO:25. This fragment may also be a fragment of Histone H3.1 or Histone H3.2, which contains an identical fragment.

The present invention may in particular be based on the use of a unique fusion protein, MCM2 HBD-G₄-TONSL ARD (SEQ ID NO: 15), allowing crystallization of the TONSL ARD-MCM2 HBD-H3-H4 tetramer complex. The fusion protein consists of the human TONSL Ankyrin Repeat Domain (ARD, residues 512-692 of SEQ ID NO:16) and MCM2 Histone-binding Domain (HBD, fragments 61-130 of SEQ ID NO:24) covalently linked through a four-Glycine linker (G₄ linker) into one expression cassette.

Useful isolated polynucleotide or amino acid sequences of the present invention is disclosed in the SEQUENCE DATA listing below having at least 90% sequence identity, such as 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 99% sequence identity or 100% sequence identity. All sequence listed are embodiments of the present invention. There are many ways to define the sequence identity of genes, nucleotides, or protein sequences. One way is through a direct comparison of two aligned sequences. Such a comparison is technically straightforward.

In particular, the term “sequence identity” as used herein may refer to the % of identical amino acids or nucleotides between a candidate sequence and a reference sequence following alignment. Thus, a candidate sequence sharing 80% amino acid identity with a reference sequence requires that, following alignment, 80% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity according to the present invention is preferably determined by aid of computer analysis, such as, without limitations, the Clustal W computer alignment program (Higgins D., Thompson J., Gibson T., Thompson J. D., Higgins D. G., Gibson T.3., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680), and the default parameters suggested therein. The Clustal W software is available from as a Clustal W WWW Service at the European Bioinformatics Institute http://www.ebi.ac.uk/clustalw. Using this program with its default settings, the candidate amino acid sequence and the reference amino acid sequence are aligned. The number of fully conserved residues are counted and divided by the length of the reference sequence. Thus, sequence identity is preferably determined over the entire length of the reference sequence. The Clustal W algorithm may similarly be used to align nucleotide sequences. Sequence identities may be calculated in a similar way as indicated for amino acid sequences. Sequence identity as provided herein is calculated over the entire length of the reference sequence.

Functional homologues of a reference peptide or polypeptide according to the invention may be peptides or polypeptides sharing at least 70%, such as at least 80%, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 98% sequence identity sequence identity with said reference peptide or polypeptide.

In another embodiment sequences that hybridize to any of the isolated polynucleotide of the present invention is disclosed in the SEQUENCE DATA listing below under conditions of low stringency, are embodiments of the present invention.

In the present context, sequence that hybridize under low stringency is defined as a sequence which specifically hybridizes to said e.g. DNA or said complementary sequence in a hybridization solution containing 0.5M sodium phosphate buffer, pH 7.2, containing 7% SDS, 1 mM EDTA at 65° C. for 16 hours and washing twice at 65° C. for twenty minutes in a washing solution containing 0.5×SSC and 0.1% SDS.

Crystallization of TONSL ARD in complex with a H4 tail or H3-H4 tetramer failed even with extensive screening. Because an additional binding protein may help to stabilize the whole complex and help crystallization, crystallization of TONSL ARD in complex with the MCM2 HBD and H3-H4 tetramer was tested.

Very tiny crystals were obtained for this complex, but failed to give big and well-diffracted crystals. The whole complex of TONSL ARD with MCM2 HBD and H3-H4 tetramer might be destabilized by the harsh crystallization conditions and form sub complexes thus hindering the optimization of the crystals.

Covalently linkage of TONSL ARD and MCM2 HBD into one cassette was thus tested through different length of Glycine linker (G_(x) linker). The G₁₂, G₁₁, G₁₀, G₉, G₈, G₇, G₆, G₅ and G₄ linkers had been tried and all these cassettes could be crystallized.

One of the constructs with a G4 linker (MCM2 HBD-G4-TONSL ARD cassette) gave well diffracted crystals in complex with H3.3(Δ56)-H4 (see Example 1), herein denoted as TONSL ARD-MCM2 HBD-H3-H4 tetramer complex (FIG. 1).

Thus, the present invention relates to a composition comprising a protein assembly of TONSL ARD-MCM2 HBD-H3-H4 in its crystalline form and/or the details of the structure of the complex deduced from structural analysis of this crystal.

Specific data defining this crystal are detailed in Table 1, Example 1. Specifically, this aspect of the invention relates to the TONSL ARD binding pocket architecture and the intermolecular interactions with the H4 tail peptide deduced from the atomic co-ordinates that define the binding surface of TONSL ARD with the histone H4 tail residues Lys12-Arg23 (e.g. in the crystal structure available in the PDB Protein Databank under the PDB ID 5JA4, DOI: 10.2210/pdb5ja4/pdb or variation thereof).

The Binding Pocket Data

The Lys12-Gly13-Gly14-Ala15 segment of H4 is positioned within a narrow surface channel of the TONSL ARD scaffold (FIGS. 2-4). The amino acid number provided in relation to histone H4 are general provided in relation to histone H4 of SEQ ID NO:34.

The intermolecular contacts spanning the Lys12-Gly13-Gly14-Ala15 segment of H4 include hydrophobic interactions between residues Gly13, Gly14 and Ala15 of H4 and residues Asn507, Cys508, Trp641, Tyr645 and Leu649 of ARD, as well as hydrogen bonds between the main-chain O of H4 Gly14 and Nε1 of ARD Trp641, and between the main-chain N of H4 Ala15 and Oη of ARD Tyr645 (FIG. 3, 8).

The main-chain O of H4 Lys16 hydrogen bonds with the Nδ2 of ARD Asn571, while the side-chain of H4 Lys16 forms contacts with ARD Asn607 and electrostatic interactions with the side-chain of ARD Glu597 (FIG. 3).

The side-chain of H4 Arg17 stacks over the side-chains of ARD Tyr572 and Cys608, while its Nη1 atom forms two hydrogen bonds with main-chain O and Oδ1 of ARD Asn571 (FIGS. 3, 5).

The side-chain of H4 H18 penetrates into a pocket lined by four strictly conserved residues (Trp563, Glu568, Asn571 and Asp604) and is positioned over His567 of ARD. The side chain of H4 His18 is stacked between Trp563 and Asn571 and forms hydrogen bonds to Glu568 and Asp604 of ARD (FIGS. 3, 6).

The main-chain O of H4 Arg19 forms a hydrogen bond with Nε1 of Trp563 and its side-chain forms contacts with Cys561 and Gly595 of ARD (FIG. 3).

The H4 Lys20 residue is bound within an acidic surface pocket on ARD adjacent to the H4 His18 binding pocket. The side-chain of H4 Lys20 interacts with the side-chain of Met528 and contacts the edge of Trp563 of ARD, while the main-chain atoms of H4 Lys20 packs against Cys561 of ARD. The Nζ atom of H4 Lys20 forms three strong hydrogen bonds (distance<3 Å) with the side-chains of strictly conserved residues Glu530, Asp559 and Glu568 of ARD, which surround H4 Lys20 within a regular triangle-like alignment (FIGS. 3, 7).

The intermolecular contacts spanning the Val21-Leu22-Arg23 segment of H4 include contacts between side-chains of H4 Val21 with Tyr560 and Cys561 of ARD, while H4 Leu22 interacts with Asp527 and Met528 of ARD. The main-chain N of H4 Arg23 forms a hydrogen bond with the main-chain O of Asp527 of ARD, while the side-chain packs against the side-chain of Tyr560 of ARD (FIGS. 3, 9).

From the structure descriptions above, it is apparent that the H4 tail (residues 12-23) with an extended β-strand like conformation lies in an elongated channel on the concave surface of the TONSL ARD. This channel is primary acidic, which provides an electrostatic complementary fit with the positively charged H4 tail. Most importantly, the side chains of H4 His18 and Lys20 are accommodated within two adjacent pockets (FIG. 4).

Substitution of H4 His18 with the larger Trp residue totally disrupts binding with TONSL ARD (FIG. 13), underscoring the importance of fitting His18 in the pocket. As the Nζ atom of H4 Lys20 forms three strong hydrogen bonds within its binding pocket, it can be predicted that methylation on H4K20 should break these critical interactions.

Both isothermal titration calorimetry (ITC) and H4 tail peptide pull-downs of recombinant ARD and full length TONSL from cell extracts confirmed that H4K20me1/2 is incompatible with TONSL binding (FIGS. 13, 14, 15, 17, 18, 19).

Further, in vitro pull-down of full-length recombinant TONSL-MMS22L with modified reconstituted mononucleosomes showed that H4K20me2 significantly reduced TONSL-MMS22L binding (FIG. 16).

The term “H4 tail binding surface of TONSL ARD” as used herein refers to the part of TONSL ARD binding the H4 tail. Amino acids of TONSL ARD involved in the binding of the H4 tail are described in detail above in this section, and the “H4 tail binding surface of TONSL ARD” may be comprise any of these amino acids. In particular, the “H4 tail binding surface of TONSL ARD” may comprise amino acids Asp527, Met528, Glu530, Asp559, Tyr560, Cys561, Trp563, Glu568, Asn571, Tyr572, Gly595, Glu597, Asp604, Asn607, Cys608, Trp641, Tyr645 and Leu649 of SEQ ID NO:16.

Our data establish that the methylation of H4K20 hinders binding with TONSL ARD. A previous study found that the ARD of the G9a/GLP methyltransferases specifically recognize H3K9me1/2, but the TONSL ARD showed no binding to H3K9me1 peptides.

Consistent with the structural data, histone H4 mutations H18A and K20A disrupted binding to TONSL in cell extracts (FIG. 11). Conversely, mutation of 6 conserved TONSL residues lining the H4 His18 and Lys20 binding pockets disrupted binding to H3-H4 and MCM2, both in vivo and in vitro without affecting binding to MMS22L, previously shown to bind to the C-terminal part of TONSL (FIGS. 10, 12). In vivo, these mutants abrogated binding to soluble H3-H4 and, consequentially, association with ASF1 and MCM2 was lost without affecting MMS22L binding to the C-terminal part of TONSL(FIG. 12).

Taken together, these results show that TONSL binds to free histones and nucleosomes via ARD recognition of H4 tails, with the important residues His18 and Lys20 fitting within two adjacent pockets. It is also shown that aa 9 to 25 of SEQ ID NO:23 can bind TONSL ARD (aa512-692 of SEQ ID NO:16), but that the mutants are strongly impaired in H4 tail peptide binding.

It is apparent that a small molecule that could bind to the His18-binding pocket or the Lys20-binding pocket, as well as either single or covalently-linked small molecules that target both pockets on TONSL ARD, would disrupt the interactions between H4 and TONSL.

Drug Design

In the most basic sense, the drug design of the present invention involves the design of single or linked small molecules that are complementary in shape and charge to the molecular target of the present invention. The invention also relates to peptides or polypeptides designed to bind to the target.

The drug design of the present invention relies on the knowledge of the three-dimensional structure presented herein. In addition to small molecules, biopharmaceuticals are an increasingly important class of drugs and computational methods for improving the affinity, selectivity, and stability of these protein-based therapeutics are also embodiments of the present invention.

The small molecules of the present invention are preferably designed so as not to affect any other important “off-target” molecules (often referred to as anti-targets), since drug interactions with off-target molecules may lead to undesirable side effects.

The term “small molecule” as used herein refers to any molecule with a molecular weight below 1000 Da, for example below 500 Da. Preferably, the “small molecule” may be an organic molecule having a molecular weight below 500 Da.

Another class of small molecules that constitute an embodiment of the invention are aptamers, which can be made up of DNA, RNA, or peptide units. Aptamers bind to a specific target molecule, and may be designed as described herein in this section. Alternatively, they may be created by selecting them from a large random sequence pool. Thus, aptamer may be any aptamer having the binding properties described herein.

In contrast to traditional methods of drug discovery (known as forward pharmacology), which rely on trial-and-error testing of chemical substances on cultured cells or animals, and matching the apparent effects to treatments, rational drug design (also called reverse pharmacology) begins with a hypothesis that modulation of a specific biological target may have therapeutic value.

In order for a small molecule to be selected as a drug target according to the present invention, one essential piece of information is required, namely the evidence that modulation of the target by the small molecule of the present invention will be disease modifying. This knowledge comes from disease linkage studies that show an association between e.g. mutations in the biological target and certain disease states.

The search for small molecules may begin by screening libraries of potential drug compounds. This may be done by using a virtual screen of existing and available small molecule libraries. The atomic co-ordinate data of the present invention or data derivable therefrom are useful in selecting and/or designing small molecules capable of interfering with the adjacent histone H4H18 and H4K20 binding pockets on the surface of the Ankyrin repeats of TONSL.

Thus, in one aspect, the present invention relates to methods for selecting and/or designing such small molecules, wherein for e.g. the method involves the use of a computer modelling or a computer program to model all or part of the structure disclosed in the PDB Protein Databank under the PDB ID 5JA4, DOI: 10.2210/pdb5ja4/pdb or data derivable therefrom, identifying a potential small molecule based on its likely ability to interact with the modelled structure, synthesising or obtaining the small molecule from a commercial source and carrying out in vitro testing of the functionality.

In one embodiment, the present invention relates to a computer-based method for identifying a small molecule capable of interfering with the adjacent histone H4H18 and H4K20 binding pocket on the surface of the Ankyrin repeats of TONSL, comprising the steps of

-   -   a) providing a 3D structural representation of the histone H4H18         and H4K20 binding pocket on the surface of the Ankyrin repeats         of TONSL in a storage medium on a computer, wherein the 3D         structural representation is derived from the atomic         co-ordinates available in the PDB Protein Databank under the PDB         ID 5JA4, DOI: 10.2210/pdb5ja4/pdb or a variant thereof in which         the r.m.s. deviation of the x, y and z co-ordinates for all         heavy atoms is less than 1.0 Å, and     -   b) using the computer to apply structure-based drug design         techniques to the structural representation.

In one embodiment, the structure-based drug design includes one or more steps of docking, such as but not limited to docking steps which screens members of a structural library.

In another embodiment, the invention relates to a computer-based method for identifying a small molecule capable of interfering with the adjacent histone H4H18 and H4K20 binding pocket on the surface of the Ankyrin repeats of TONSL, comprising the steps of

-   -   a) providing a 3D structural representation of the histone H4H18         and H4K20 binding pocket on the surface of the Ankyrin repeats         of TONSL in a storage medium on a computer, wherein the 3D         structure! representation is derived from the atomic         co-ordinates of available in the PDB Protein Databank under the         PDB ID 5JA4, DOI: 10.2210/pdb5ja4/pdb or a variant thereof in         which the r.m.s. deviation of the x, y and z co-ordinates for         all heavy atoms is less than 1.0 Å, and     -   b) using the computer to apply structure-based drug design         techniques to the structural representation, and     -   c) providing a compound identified by said structure based drug         design technique, and     -   d) contacting said compound with the binding pocket on the         surface of the Ankyrin repeats of TONSL and assaying the         interaction between them.

In another embodiment, the invention relates to method of identifying a small molecule capable of interfering with the histone H4H18 and H4K18 binding pocket on the surface of the Ankyrin repeats of TONSL, or a fragment or variant thereof, said method comprising the steps of:

-   -   a. generating the spatial structure of the pocket on a computer         screen using atomic coordinates as presented in the PDB Protein         Databank under the PDB ID 5JA4, DOI: 10.2210/pdb5ja4/pdb, data         derivable therefrom, or by a root mean square deviation over         protein backbone atoms of not more than 1.0 Å,     -   b. generating potential small molecules with their spatial         structure on the computer screen, and     -   c. selecting small molecules that can bind to at least one amino         acid residue of the set of binding interaction sites.

In another embodiment, the invention relates to a computer-assisted method for identifying a small molecule capable of interfering with the histone H4H18 and H4K20 binding pocket on the surface of the Ankyrin repeats of TONSL, or a fragment or variant thereof, using a programmed computer comprising a processor, a data storage system, a data input device and a data output device, comprising the following steps:

-   -   a. inputting into the programmed computer through said input         device data comprising; atomic coordinates of a subset of the         atoms according to the present invention, thereby generating a         criteria data set; wherein said atomic coordinates are selected         from the atomic coordinates as presented in the PDB Protein         Databank under the PDB ID 5JA4, DOI: 10.2210/pdb5ja4/pdb, data         derivable therefrom, or by a root mean square deviation over         protein backbone atoms of not more than 1.0 Å,     -   b. comparing, using said processor, the criteria data set to a         computer data base of low-molecular weight organic chemical         structures and peptide fragments stored in the data storage         system, and     -   c. selecting from said data base, using computer methods, a         chemical structure having a portion that is structurally         complementary to the criteria data set.

In another embodiment, the invention relates to a method for identifying a ligand, comprising the steps of:

-   -   a. selecting a potential ligand using atomic coordinates in         conjunction with computer modelling, wherein said atomic         coordinates are the atomic coordinates as presented in the PDB         Protein Databank under the PDB ID 5JA4, DOI:         10.2210/pdb5ja4/pdb, data derivable therefrom, or by a root mean         square deviation over protein backbone atoms of not more than         1.0 Å, by docking potential ligands into a set of binding         interaction sites, said binding interaction generated by         computer modelling and selecting a potential ligand capable of         binding to at least one amino acid in said set of binding         interaction sites of TONSL,     -   b. providing said potential ligand and said TONSL,     -   c. contacting the potential ligand with said TONSL, and     -   d. detecting binding of said potential ligand with TONSL.

A non-limiting Example of methods for identifying small molecules or inhibitors according to the invention is provided in Example 16 herein below.

SCHRODINGER Analysis

The small molecule drug discovery suite by SCHRODINGER contains a comprehensive set of programs aimed at state-of-the-art support of every step of drug discovery and optimization. This represents one type of structure-based docking programs for small molecule inhibitor design, other could also be applied. The essence of drug discovery is in (1) finding key adjacent pocket(s) on a target protein that affects or controls its function and (2) finding covalently linked small molecules with high-affinity binding to the adjacent pocket(s) in the known databases followed by iterative optimization through addition/deletion of substituents while maximizing shape, hydrogen bonding and electrostatic complementarity.

The most effective definition of a binding pocket is possible when a crystal structure of a protein-ligand complex is known. In this case, the high precision GLIDE algorithm is applied to calculate the grid, which is subsequently used for high-throughput docking of commercially available small molecules. This grid represents the three-dimensional spatial information about essential components of binding surfaces such as all lipophilic atoms, all hydrogen-bonding atoms with a score accounting for orientation of hydrogen bonds, metal atoms if present in the protein, distribution of charges, as well as van der Waals atomic interactions. The ligand conformations are also pre-computed in SCHRODINGER through application of LigPrep procedure that filters out the least probable configurations and accounts for various pH-dependent ionization states of the prospective ligands.

Docking computations for two interacting components thus represented are highly precise and can be performed on high scale and with various degrees of controlled flexibility within the protein component. The protein-ligand complexes with ligands screened from databases can next be ranked using advantages of post-docking Embrace minimizations and prime MM GBSA of the SCHRODINGER suite. These procedures give estimates of free binding energy and thus are suitable tools for ligand ranking based on the computed binding affinity.

When the crystal structure of a drug-protein complex is not known, as is the case in TONSL-H4 tail complex, the protein binding pocket(s) and corresponding grid(s) can be computed from the assumption that binding of the prospective ligand(s) should outcompete the most critical aspects for interacting such as for e.g. amino acid residues H18 and K20 on histone H4 tail or any of the above listed interactions.

In this case, the docking grid(s) are computed for the TONSL pocket(s) as defined by the cavities that incorporate these two residues: individually or through being covalently connected together. The scoring function for the competing small molecules in this case is compared with that of the original binding targets. Optimization of identified small molecule leads is achieved by introduction/deletion of R-groups and computing the resulting effect on their affinity by isothermal titration calorimetry, prior to chemical synthesis of the most promising candidates.

Prospective drug candidates are generated in an iterative process of x-ray structure determination and ligand optimization, with the goal of identifying covalently linked optimized ligands that simultaneously target the H4H18 and H4K20 pockets on the TONSL scaffold.

The route to effective compounds according to the present invention is to

-   -   1. Determine by the use of the structure scaffold/co-ordinates         in-silico and standard binding/scoring algorithms in-silico         molecules with high binding affinity to the TONSL/H4 crystal         structure disclosed herein     -   2. Modify the small molecule structures (with R-substitutions)         in silico to represent drug-like compounds     -   3. Synthesize such structures by standard chemistry     -   4. Test the resulting synthesized structures in the assays of         the invention     -   5. Select, modify and optimize the most promising compounds.

An alternative to this approach is to screen existing libraries in the assays, and optionally optimize these so as bind in the space as described.

Thus, in one embodiment the present invention relates to a method of selecting or designing a small molecule capable of interfering with the histone H4H18 and H4K20 binding pocket on the surface of the Ankyrin repeats of TONSL, said method comprises use of at least part of the atomic co-ordinates data contained in the PDB Protein Databank under the PDB ID 5JA4, DOI: 10.2210/pdb5ja4/pdb or data derivable therefrom, wherein said method involves use of a computer modelling package or a computer program to model all or part of the structure of MCM2 HBD-G4-TONSL ARD in complex with H3 (57-135) and H4, identifying such small molecule based on its likely ability to prevent or disrupt the H4 tail with the Ankyrin repeats of TONSL in the modelled structure.

In one embodiment, the invention relates to a fusion protein, MCM2 HBD-G₄-TONSL ARD (SEQ ID NO: 15) or variants thereof (e.g. G_(x) linker =G₁₂, G₁₁, G₁₀, G₉, G₈, G₇, G₆, or G₅), which are instrumental for determining the structural details of TONSL binding small molecule inhibitors.

In another embodiment, the invention relates to the TONSL and H4 mutants that disrupt interaction between TONLS and TONSL ARD with histone H4 (FIGS. 10-13, Examples 2-4). The mutant data identify key intermolecular interactions contributing to the specificity and stability of complex formation. The verified TONSL histone-binding mutants are instrumental for biochemical and biological assay design to assess the efficiency of screened small molecule inhibitors in blocking TONSL-histone interaction and TONSL function.

In one embodiment the invention relates to compounds having a 3-[(3-Aminocyclopentyl)carbonyl]-1H-quinolin-4-one core. Such compounds may be any compound, preferably any small organic compound comprising the 3-[(3-Aminocyclopentyl)carbonyl]-1H-quinolin-4-one, wherein one or more positions may be substituted with a substituent. Thus, the compound may be a 3-[(3-Aminocyclopentyl)carbonyl]-1H-quinolin-4-one, wherein one or more —H have been substituted for another group.

Peptide Inhibitors of TONSL

The invention also provides inhibitors of TONSL, in particular such inhibitors, which can inhibit binding of TONSL ARD to histone H4. In preferred embodiments the inhibitor may be a compound binding the histone H4 tail binding surface of TONSL ARD.

Methods for identifying such inhibitors are provided herein elsewhere.

The inhibitor may be any useful compound for example a peptide inhibitor or a small molecule. In one embodiment of the invention, the inhibitor is a peptide inhibitor. The peptide inhibitor may for example be useful in the treatment of cancer.

As used herein the term “peptide inhibitor” refers to a compound comprising a peptide or a polypeptide optionally linked to a conjugated moiety. The peptide or polypeptide part of the peptide inhibitor is in the following referred to as “peptide”.

The peptide inhibitor may thus be a peptide, which is capable of binding the TONSL ARD. Thus, the peptide inhibitors may be any of the peptides described herein below, wherein said peptide is capable of binding to TONSL ARD (e.g. to a peptide consisting of amino acids 512 to 692 of SEQ ID NO:16) with a Kd of at the most 10 μM, preferably a Kd of at the most 5 μM, such as with a Kd of at the most 3 μM. Said Kd may for example be determined as described in Example 10 and 15 below.

In one embodiment the peptide may comprise the sequence motif I: Arg-His-Xaa-Lys (SEQ ID NO:26), wherein Xaa may be any amino acid.

In one embodiment the peptide may comprise the sequence motif II: Arg-His-Xaa-Lys-Val-Leu (SEQ ID NO:27), wherein Xaa may be any amino acid.

In one embodiment the peptide may comprise the sequence motif III: Val-Leu-Arg.

In one embodiment the peptide may comprise the sequence motif IV: Arg-His-Xaa-Lys-Val-Leu-Arg (SEQ ID NO:28), wherein Xaa may be any amino acid.

In particular, the peptide may be a peptide consisting of in the range of 4 to 40 amino acids comprising the sequence Arg-His-Xaa-Lys, and/or one or more of the sequence motifs I, II, III or IV. The peptide may also be a peptide consisting of in the range of 4 to 25 amino acids, such as in the range of 4 to 15 amino acids, preferably in the range of 7 to 15 amino acids, such as in the range of 7 to 12 amino acids, such as in the range of 4 to 10 amino acids, for example in the range of 6 to 9 amino acids comprising the sequence Arg-His-Xaa-Lys, and/or one or more of the sequence motifs I, II, III or IV. The peptide may also be a peptide consisting of at the most 25 amino acids, preferably at the most 15 amino acids, such as at the most 12 amino acids, for example at the most 9 amino acids, wherein the peptide comprises one or more of the sequence motifs I, II, III or IV. Said Lys is preferably unmethylated. Said Xaa may be any amino acid, for example it may be selected from the group consisting of Ala and Arg. In one embodiment of the invention Xaa is not Arg.

In one embodiment the peptide comprises or consists of:

-   -   I. a sequence consisting of amino acid 12 to 23 of SEQ ID NO:23         or of SEQ ID NO:34; or     -   II. a functional homologue thereof consisting of a sequence of         amino acid 12 to 23 of SEQ ID NO:23 or of SEQ ID NO:34, wherein         up to 5 amino acids, for example up to 4 amino acids, for         example up to 3 amino acids, such as up to 2 amino acids, for         example 1 amino acids may be substituted, and wherein said         peptide comprises at least Arg17, His18 and Lys20 of SEQ ID         NO:23 or of SEQ ID NO:34,     -   III. a functional homologue thereof consisting of a sequence of         amino acid 12 to 23 of SEQ ID NO:23, wherein up to 6 amino         acids, such as up to 5 amino acids, for example up to 4 amino         acids, such as up to 3 amino acids, for example up to 2 amino         acids may be substituted may be substituted,

with the proviso that the inhibitor is different to histone H4 of SEQ ID NO:23.

In one embodiment the peptide comprises or consists of:

-   -   IV. a sequence consisting of amino acid 9 to 25 of SEQ ID NO:23         or of SEQ ID NO:34; or     -   V. a functional homologue thereof consisting of a sequence of         amino acid 9 to 25 of SEQ ID NO:23 or of SEQ ID NO:34, wherein         up to 5 amino acids, for example up to 4 amino acids, for         example up to 3 amino acids, such as up to 2 amino acids, for         example 1 amino acids may be substituted, and wherein said         peptide comprises at least Arg17, His18 and Lys20 of SEQ ID         NO:23 or of SEQ ID NO:34,     -   VI. a functional homologue thereof consisting of a sequence of         amino acid 9 to 25 of SEQ ID NO:23 or of SEQ ID NO:34, wherein         up to 6 amino acids, such as up to 5 amino acids, for example up         to 4 amino acids, such as up to 3 amino acids, for example up to         2 amino acids may be substituted

with the proviso that the inhibitor is different to histone H4 of SEQ ID NO:23.

In one embodiment the peptide comprises or consists of:

-   -   VII. a sequence consisting of amino acid 14 to 33 of SEQ ID         NO:23 or of SEQ ID NO:34; or     -   VIII. a functional homologue thereof consisting of a sequence of         amino acid 14 to 33 of SEQ ID NO:23 or of SEQ ID NO:34, wherein         up to 5 amino acids, for example up to 4 amino acids, for         example up to 3 amino acids, such as up to 2 amino acids, for         example 1 amino acids may be substituted, and wherein said         peptide comprises at least Arg17, His18 and Lys20 of SEQ ID         NO:23,     -   IX. a functional homologue thereof consisting of a sequence of         amino acid 14 to 33 of SEQ ID NO:23 or of SEQ ID NO:34, wherein         up to 6 amino acids, such as up to 5 amino acids, for example up         to 4 amino acids, such as up to 3 amino acids, for example up to         2 amino acids may be substituted

with the proviso that the inhibitor is different to histone H4 of SEQ ID NO:23.

In embodiments of the invention where the peptide comprises a functional homologue as described under III., VI. or IX herein above it may be preferred that:

-   -   I. the amino acid corresponding to amino acid 17 of SEQ ID NO:23         or of SEQ ID NO:34 is a charged amino acid, such as a positively         charged amino acid, for example an amino acid selected from the         group consisting of Lys and Arg     -   II. the amino acid corresponding to amino acid 18 of SEQ ID         NO:23 or of SEQ ID NO:34 is a charged amino acid, such as a         positively charged amino acid, for example His     -   III. the amino acid corresponding to amino acid 20 of SEQ ID         NO:23 or of SEQ ID NO:34 is a charged amino acid, such as a         positively charged amino acid, for example an amino acid         selected from the group consisting of Lys and Arg.

In one embodiment, the peptide inhibitors may be identified based on the sequence of histone H4 of SEQ ID NO:23 or a fragment thereof, i.e. by systematically or randomly replacing one or more amino acids of an H4 peptide, or certain parts of H4 native peptide, with other amino acids.

In general the peptide inhibitor should not be too large. Accordingly, it may be preferred that the peptide consists of at the most 40 amino acids, such as at the most 25 amino acids, for example at the most 20 amino acids, such as at the most 15 amino acids, for example at the most 12 amino acids. For example, the peptide may comprise or consist of in the range of 4 to 40, for example in the range of 4 to 25, for example in the range of 4 to 20, such as in the range of 4 to 15, such as in the range of 4 to 12, for example in the range of 7 to 15 amino acids, such as in the range of 7 to 12 amino acids, such as in the range of 5 to 10 or such as in the range of 6 to 9 consecutive amino acids of SEQ ID NO:23.

In one embodiment, the peptide may comprise or consist of in the range of 4 to 40, for example in the range of 4 to 25, for example in the range of 4 to 20, such as in the range of 4 to 15, such as in the range of 4 to 12, for example in the range of 7 to 15 amino acids, such as in the range of 7 to 12 amino acids, such as in the range of 5 to 10 or such as in the range of 6 to 9 consecutive amino acids of SEQ ID NO:23, wherein up to 3, such as up to 2, for example at the most one amino acid may have been substituted for another amino acid, and wherein the peptide comprises one or more of the sequence motifs I, II, III or IV.

Said functional homologues of fragments of SEQ ID NO:23 are preferably peptides, comprising above defined sequence, and which are capable of binding TONSL.

As described above the peptide may comprise one or more Lys residues. It may be preferred that at least one Lys residue, for example all Lys residues are unmethylated. In particular, when the peptide comprises a fragment of SEQ ID NO:23 it may be preferred that the amino acid corresponding to Lys20 of SEQ ID NO:23 is unmethylated.

In some embodiments of the invention, the C-terminal of the peptide is amidated, i.e. having the chemical structure —C(O)NH₂.

In some embodiments of the invention, the C-terminal of the peptide is alkylated e.g methylated i.e. having the chemical structure —C(O)OCH₃.

In some embodiments of the invention, the N-terminal of the peptide is acetylated i.e. having the chemical structure CH₃C(O)N(H)—.

In some embodiments of the invention, the N-terminal of the peptide is formylated i.e. having the chemical structure HC(O)N(H)—.

It is understood that a peptide consisting of a given sequence may have a C-terminal, which is amidated or alkylated and/or an N-terminal, which is acetylated or formylated.

The peptide inhibitor may in particular be a peptide comprising or consisting of a peptide selected from the group consisting of:

Ala-Lys-Arg-His-Arg-Lys-Val-Leu-Arg-NH₂, Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-Leu-Arg-NH₂, Lys-Gly-Gly-Ala-Lys-Arg-His-Ala-Lys-Val-Leu-Arg-NH₂ and Lys-Gly-Gly-Ala-Ala-Arg-His-Arg-Lys-Val-Leu-Arg-NH₂.

The moiety “—NH₂” indicated in the sequence above indicates that the C-terminal of the peptides are amidated. The invention however also encompass peptides which are not amidated. Accordingly, in one embodiment, the invention relates to peptide inhibitors comprising or consisting of at the most 40, for example at the most 25, for example at the most 20, such as at the most 15, such as at the most 12 amino acids, wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33.

In some embodiments of the invention, the peptide is a TONSL mutant polypeptide, for example any of the TONSL mutant polypeptides described herein below in the section “TONSL mutant polypeptides”. For example, the peptide may be selected from the group consisting of polypeptides of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22.

In some embodiments of the invention the inhibitor is a fragment of TONSL, in particular a fragment of TONSL capable of binding histone H4, but incapable of binding at least one other binding partner of TONSL, e.g. incapable of binding MMS22L. Said fragment of TONSL, may for example be a fragment comprising the H4 tail binding surface of TONSL. In one embodiment, the fragment comprises or consists of amino acids 512 to 692 of SEQ ID NO:16 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity therewith.

The peptide described herein may in some embodiments be linked to a conjugated moiety, for example the peptide may be covalently linked to a conjugated moiety. Said conjugated moiety may for example be any of the moieties described herein below.

In one embodiment the conjugated moiety may be a peptide, a sugar, a lipid, a polymeric molecule or any other chemical group that can be covalently linked to a peptide. For example, the conjugated moiety may improve the physical properties of the peptide, such as its solubility, stability or half-life.

In one embodiment the conjugated moiety is a polymeric molecule, such as polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP). The polymeric molecule may also be a modified PEG, for example NPEG.

Assays Validating the Small Molecules of the Present Invention

Molecules identified according to the present invention are tested in biochemical and cell biology assays listed below. Molecules that show activity in these assays towards inhibiting TONSL function similar to TONSL ARD mutants are further tested in high-throughput biological assay for efficacy in killing cancer cells and ultimately in pre-clinical and clinical trials.

Biochemical Screening and Biophysical Testing (Assays)

The Key Components in the Assays are:

-   -   a) Recombinant human TONSL ARD (residues 512-692, point 1)     -   b) Recombinant human TONSL ARD mutants E530A, D559A, W563A,         E568A, N571A, D604A (point 2)     -   c) Histone H4 peptide-containing K20me0 with or without         acetylation at K16 (H4 tail covering the critical residues for         TONSL binding, for example 9-25 or 14-33) coupled to fluorescent         molecule or biotin     -   d) Histone H4 peptide containing K2Ome2 with or without         acetylation at K16 as a control for no binding to TONSL ARD H4         tail covering the critical residues for TONSL binding, for         example 9-25 or 14-33) coupled to fluorescent molecule or biotin     -   e) Human cells expressing tagged TONSL ARD wild type (WT) or         histone-binding mutant (e.g. any of the mutants described herein         in the section “TONSL mutant polypeptide”, for example any of         E530A, D559A, W563A, E568A, N571A, or D604A,). We generated and         verified expression constructs for GFP-fusion proteins for         mutants and inducible cell lines for WT, E568A and N571A

Assay 1: High-throughput (HT) Measurements of Small Molecule Dependent Disruption of the TONSL ARD-H4 Tail Interaction Detailed in Point 2.

First line screening may be based on Fluorescence anisotropy measurements using histone H4 peptides conjugated with a fluorescent probe. Assay conditions are as detailed below in our demonstrated ITC assay (Example 2), with the critical criteria that ARD binding being observed to H4 (residues 9-25), but not H4K20me2 (residues 9-25, dimethylated at K20) (FIG. 13).

Assay 2: Measurement of Binding Properties of Small Molecule Inhibitors to ARD

Second line screening may involve Isothermal titration calorimetry (ITC) to determine binding affinity/association constants (Ka) and Surface plasmon resonance (SPR) assays to determine rate constants. The resulting molecules yielding higher binding affinity to TONSL than unmodified histone H4 peptide (Example 2) are further shortlisted to identify those with μM to nM effective range.

Assay 3. Crystallization and Optimization

Candidate small molecules may be crystallized with TONSL ARD (using our demonstrated crystallization conditions outlined in point 1, Example 1), providing further possibility for drug optimization.

Assay 4. Competition Assay Based on Peptide Pulldown

a. Selected small molecules are validated for their ability to compete out binding of recombinant ARD to H4K2Ome0 peptides (H4K2Ome2 and ARD mutants are used as negative controls, assay conditions as in Example 3).

b. Selected small molecules are validated for their ability to compete out binding of GFP-TONSL-MMS22L to H4K2Ome0 peptides in cell extracts.

Grow cells expressing tagged TONSL WT or ARD mutant (as control for no histone H4 binding). Prepare whole-cell extracts according to standard protocols (documented conditions for this assay in Example 4). Incubate the cell extracts with surface-bound biotinylated-histone H4 peptides (e.g. H4K2Ome0 vs. H4K2Ome2 as a control for no binding to WT TONSL). Dose-response analysis with small molecule inhibitors assaying loss of WT TONSL binding to H4K2Ome0 peptides by ELISA, western blotting or similar approaches.

HT Biological Assays to Screen for Small Molecule Inhibitors

The present inventors have demonstrated that TONSL binds to chromatin via ARD recognition of the tails of nucleosomal histone H4 unmethylated at K20 (FIG. 21). The biological function of a small molecule inhibitor is thus to prevent TONSL from binding histone H4 and displace TONSL-MMS22L complex from chromatin.

The present inventors have developed high-content microscopy based assays, which are used for drug screening (Example 5).

-   -   1) Immunofluorescence staining of endogenous TONSL shows binding         to post-replicative chromatin, which is lost upon depletion of         TONSL with RNAi (FIG. 20).     -   2) binding of GFP-TONSL to chromatin is disrupted by ARD mutants         that disrupt H4 binding (FIG. 21).

The present invention covers small molecule inhibitors that displace TONSL and TONSL-MMS22L complex from post-replicative chromatin and mimic TONSL ARD mutants.

Key Components:

-   -   a) Anti-TONSL antibody (Sigma, ref. nr. HPA0244679), tested in         our high-content assay (Example 5)     -   b) Pre-extraction conditions to remove soluble TONSL (Example 5)     -   c) Condition for TONSL depletion (negative control) (Example 5)     -   d) GFP-TONSL WT and ARD mutant cell lines

Rationale of HT Assays:

-   -   1. High-content imaging to identify small molecules that remove         endogenous TONSL and TONSL-MMS22L complex from chromatin (FIG.         21)     -   2. High-content imaging of TONSL binding to chromatin using         cells expressing GFP-TONSL WT and ARD mutant (N571A, point 2)         upon incubation with TONSL inhibitors by high-content imaging

Assay 1: HT Imaging of Chromatin-bound Endogenous TONSL

Cells are grown in a HT format (96 or 384 well-plates). Incubated with small molecule TONSL inhibitors in a dose-response and time-course set-up. Soluble proteins are removed by cell pre-extraction followed by cell fixation according to standard protocols. Detect chromatin-bound TONSL with commercial antibody (Sigma, ref. nr. HPA0244679) documented by us for specificity (Example 5). TONSL siRNA are used as control. Chromatin-bound TONSL may be quantified by conventional methods, for example using standard high content imaging (Example 5) or alternatively by FACS analysis.

Assay 2: HT Imaging of GFP-TONSL

Grow cells with expression of GFP-TONSL WT and ARD mutant (N571A, point 2: negative control for binding) in a HT format (96 or384 well-plates). Incubate with small molecule TONSL inhibitors in a dose-response and time-course set-up. Remove soluble proteins by pre-extraction cells and fix cells according to standard protocols. Quantify GFP-TONSL levels on chromatin by standard high content imaging (Example 5).

Biological Assays to Measure HR Inhibition

TONSL is required for HR of damaged replication forks and DNA double strand breaks (Duro et al. 2010 Mol Cell 40:619; O'Donnell et al. 2010 Mol Cel 40:619; O'Connell et al. 2010 Mol Cell 40:645; Piwko et al. EMBO J 2010 29:4210). Thus, small molecule inhibitors preventing TONSL or MMS22L recruitment to chromatin phenocopy the loss of TONSL or MMS22L.

To test the capacity of small molecule inhibitors to compromise HR, published assays to monitor recruitment of repair proteins (e.g., Duro et al. 2010 Mol Cell 40:619; O'Donnell et al. 2010 Mol Cel 40:619; O'Connell et al. 2010 Mol Cell 40:645; Piwko et al. EMBO J 2010 29:4210) and HR reporter cell lines are used (e.g., Pierce et al., Genes Dev 13:2633, 1999). In those assays, the cells are treated with small molecule TONSL inhibitors in a time and dose-dependent manner and efficiency of HR repair is assayed by high-content microscopy

TONSL Mutant Polypeptide

In one embodiment the invention relates to a TONSL mutant polypeptide. Said TONSL mutant polypeptide is preferably a TONSL mutant polypeptide, which is incapable of binding histone H4, but which retains other TONSL functions. Preferably, said TONSL mutant polypeptide is capable of binding MMS22L and wherein said TONSL mutant does not bind histone H4. Such TONSL mutant polypeptides will function as dominant negative mutants, because they will bind MMS22L and other members of the complex, preferably with the same or similar affinity as the wild type TONSL polypeptide, but they will not bind the post-replicative chromatin to any significant extent.

In one embodiment the TONSL mutant polypeptide is a polypeptide of SEQ ID NO:16 carrying one or more mutations, preferably one or more mutations in amino acid(s) contributing to the H4 tail binding surface of TONSL ARD. Thus, the TONSL mutant polypeptide may comprise at least one mutation in an amino acid of the histone H4 tail binding surface of the TONSL ARD, wherein said TONSL mutant polypeptide apart from said mutation is identical to SEQ ID NO:16 or shares a sequence identity with SEQ ID NO:16 of at least 70%, wherein said TONSL mutant polypeptide is capable of binding MMS22L and wherein said TONSL mutant does not bind histone H4. In particular said TONSL mutant polypeptide may be a polypeptide of SEQ ID NO:16 carrying a mutation in at least one amino acid selected from the group consisting of amino acid number 527, 528, 530, 559, 560, 561, 563, 568, 571, 572, 595, 597, 604, 607, 608, 641, 645 and 649 of SEQ ID NO:16.

In one embodiment the TONSL mutant polypeptide carries a mutation in one or more of the amino acids selected from the group consisting of: Glu530, Asp559, Trp563, Glu568, GIn571 and D604 of SEQ ID NO:16.

In one embodiment TONSL mutant polypeptide may comprise or even consist of a sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22.

The TONSL mutant polypeptide may be linked to a conjugated moiety, for example the TONSL mutant polypeptide may be linked to a detectable marker. Said detectable marker may be a peptide or a polypeptide, e.g. a fluorescent protein, such as GFP.

A Method for Predicting the Effect of Inhibition of TONSL

The invention also provides methods for predicting the effect of inhibition of TONSL. As described herein above, the TONSL mutant polypeptides described herein may be dominant negative mutant. Expression of such mutants may phenocopy TONSL/MMS22L depletion, and thus mimic the actions of an inhibitor of TONSL binding to histone H4.

Thus, in one embodiment the invention relates to methods for predicting the effect of inhibition of TONSL, said method comprising the steps of

-   -   expressing a TONSL mutant polypeptide in an organism and/or         cells of an organism, wherein said polypeptide optionally is         expressed conditionally,     -   determining the effect of said polypeptide in said organism         and/or cells wherein said effect of expressing said polypeptide         in said organism and/or cells indicates the effect of inhibition         of TONSL in said organism and/or cells.

In particular, said TONSL mutant polypeptide may be any of the TONSL mutant polypeptides described herein above in the section “TONSL mutant polypeptide”. The organism and/or cells may comprise a heterologous nucleic acid encoding any of said TONSL mutant polypeptides.

Thus, the effect obtained after expression of a dominant negative TONSL mutant polypeptide may mimic the effect of inhibition of TONSL in said organism and/or cells. Thus, the effect observed is indicative of the effect expected to be obtained, if the organism/cells were treated with an inhibitor of TONSL, in particular with an inhibitor inhibiting binding between TONSL ARD and the histone H4.

The organism or the cells may represent a disease model, and thus the method may be used to predict the efficacy of treating said disease with an inhibitor or TONSL, e.g. an inhibitor inhibiting binding between TONSL ARD and the histone H4. The organism may be any organism, e.g. a mammal, for example a mouse, a rat or a rabbit. The organism may be genetically modified to contract a particular disease, for example cancer. Thus, the organism may be a mouse genetically engineered to contract cancer.

The cells may also represent a disease model. The cells may be any cells, such as mammalian cells, e.g. human cells. The cells may be cultivated using any conventional method. For example, the cells may be cultured in a 3D culture, which more closely may mimic in vivo conditions.

Since expression of TONSL mutant polypeptide may be toxic to cells, it may be preferred that said TONSL mutant polypeptide is expressed only conditionally. Thus, the TONSL mutant polypeptide may be expressed only in specific cells of said organism and/or said TONSL mutant polypeptide may be induced to be expressed at specific times. Thus, the organisms and/or cells may comprise a heterologous nucleic acid encoding any of the TONSL mutant polypeptides described herein above in the section “TONSL mutant polypeptide” operably linked to an inducible promoter. In that manner expression of said TONSL mutant polypeptide may be induced at any specific time. Inducible promoters and well known in the art and the skilled person will be able to select a useful promoter. The organism may also comprise a heterologous nucleic acid encoding any of said TONSL mutant polypeptides operably linked to a promoter directing expression in only some cell types. Such promoters are also known in the art.

Method for Determining TONSL Inhibitory Effect

The invention also provides methods of identifying the TONSL inhibitory effect of a putative inhibitor of TONSL. In particular said methods can be used to determine whether a compound is capable of inhibiting binding between TONSL and histone H4. Such methods are useful for screening for inhibitors of TONSL. However, the methods are also useful for validating whether a putative inhibitor in fact is capable of inhibiting TONSL. Thus, the methods can be used for determining biosimilarity between a known

TONSL inhibitor and a similar compound, which is a putative TONSL inhibitor. The methods may also be used in quality control of TONSL inhibitors. The methods may also be used to validate the inhibitory effect of an inhibitor identified by computer aided techniques using the atomic coordinates provided in the PDB Protein Databank under the PDB ID 5JA4, DOE: 10.2210/pdb5ja4/pdb.

Said methods may comprise the steps of

-   -   a) providing a test compound, which is a putative inhibitor of         TONSL     -   b) providing an host organism expressing TONSL of SEQ ID NO:16,         TONSL ARD consisting of amino acids 512 to 692 of SEQ ID NO:16         or a functional homologue of any of the aforementioned sharing         at least 90% sequence identity to amino acids 512 to 692 of SEQ         ID NO:16;     -   c) contacting said host organism with said putative inhibitor     -   d) detecting chromatin associated TONSL, TONSL ARD or functional         homologue thereof in said host organism, wherein reduction of         chromatin associated TONSL, TONSL ARD or a functional homologue         thereof is indicative of said test compound being an inhibitor         of TONSL.

Said reduction of chromatin associated TONSL, TONSL ARD or a functional homologue thereof is preferably a reduction compared to said level in said host organism in the absence of said inhibitor. In particular, said reduction may be a reduction to less than 50%, such as a reduction to less than 30%, such as a reduction to less than 20%, for example a reduction to less than 10% of the chromatin associated TONSL, TONSL ARD or a functional homologue thereof in said host organism in the absence of said inhibitor. In one embodiment, the reduction is that there is no detectable chromatin associated TONSL, TONSL ARD or a functional homologue thereof.

The host organism may be any useful host organism including cells, e.g. mammalian cells maintained in a tissue culture. Thus, the term “cells of the host organism” may refer to part of a multicellular host organism or it may refer to the host organism per se, when the host organism is cells. The host organism may endogenously express TONSL, in which case the method may involve detecting chromatin associated endogenous TONSL. Endogenous TONSL may be detected by various means, but frequently, endogenous TONSL is detected by means of an antibody, a binding fragment of an antibody or another binding molecule specifically recognising and binding TONSL. The antibody, fragment or binding molecule may be directly labelled with a detectable label. It is also possible that the methods involve use of a secondary antibody, fragment or binding molecule binding the first antibody, fragment or binding molecule, wherein the secondary antibody, fragment or binding molecule is labelled with a detectable label. Other methods for detecting binding of an antibody, fragment or binding molecule are also available and may be used with the methods.

The detectable label may be any detectable label including but not limited to fluorophores, bioluminescents, chemoluminescents, dyes, enzymes, heavy metals, or radioactive compounds. In preferred embodiments the detectable label is a fluorophore, i.e. a fluorescent moiety.

It is also comprised within the invention that said host organism comprises a heterologous nucleic acid encoding said TONSL, TONSL ARD or functional homologue thereof. In such embodiments the methods may involve detecting chromatin associated heterologous TONSL, TONSL ARD or functional homologue thereof. The heterologous TONSL, TONSL ARD or functional homologue thereof may be linked to a detectable label, for example to a fluorescent polypeptide, such as GFP. Thus, the step of detection, may be a step of detecting the detectable label. Heterologous TONSL, TONSL ARD or functional homologue thereof may also be detected used the same methods useful for detection of endogenous TONSL.

The detectable label may be detected using any technical means useful for detecting the particular detectable label. In embodiments of the invention, where the detectable label is fluorescent, then the detectable label may be detected by means of FACS, fluorescent microscopy or high content microscopy.

Step d) of the method may comprise a step of extracting soluble proteins from the cells of said host organism. This step may ensure that all TONSL, TONSL ARD or functional homologues thereof, which is not chromatin associated is extracted from the cells of said host organism. Following extraction of soluble proteins all remaining TONSL, TONSL ARD or functional homologues thereof may be regarded as chromatin associated TONSL, TONSL ARD or functional homologues thereof.

Extracting soluble protein may be done in any conventional manner, e.g. by permeabilising cell membranes, e.g. by use of a detergent optionally followed by one or multiple washing steps. Any detergent may be used for permeabilisation, e.g. Triton (e.g. Triton X-100), NP-40, saponin, Tween (e.g. Tween-20), Digitonin or Leucoperm. After extraction the cells may be fixed prior to detection. Fixation reagents are well known in the art and includes for example aldehydes, e.g. formaldehyde, paraformaldehyde or glutaraldehyde.

One useful method for determining chromatin associated TONSL, TONSL ARD or functional homologues thereof is described in Example 5 herein. Other useful methods for determining chromatin associated TONSL, TONSL ARD or functional homologues thereof are described in the section “High-throughput (HT) biological assays for efficacy of small molecule inhibitors in killing cancer cells”, for example in Assay 1 and 2 of that section.

The methods for determining TONSL inhibitory effect may also be any of the methods described herein in the section “Assays validating the small molecules of the present invention”, for example any of the assays 1, 2, 3 or 4. Thus, these methods may not only be used for validating small molecules, but can also generally be used for determining TONSL inhibitory effect of a compound.

In one embodiment the method comprises the steps of:

-   -   a) Providing histone H4 or a fragment thereof comprising at         least amino acids 17 to 20, such as at least amino acids 12 to         23, for example at least amino acids 9 to 25, such as at least         amino acids 14 to 33 of SEQ ID NO:23, wherein said histone H4 or         fragment thereof optionally may be linked to a detectable label;     -   b) providing TONSL of SEQ ID NO:16, TONSL ARD consisting of         amino acids 512 to 692 of SEQ ID NO:16 or a functional homologue         of any of the aforementioned sharing at least 90% sequence         identity to amino acids 512 to 692 of SEQ ID NO:16     -   c) Incubating said histone H4 or fragment thereof with said         TONSL, TONSL ARD or functional homologue thereof in the presence         of a putative inhibitor of TONSL     -   d) Determining whether binding between said histone H4 or         fragment thereof with said TONSL, TONSL ARD or functional         homologue thereof     -   wherein reduction in binding of said histone H4 or fragment         thereof with said TONSL, TONSL ARD or functional homologue         thereof is indicative of that said putative inhibitor of TONSL         is capable of inhibiting TONSL.

Said reduction in binding may in particular be a reduction to at the most 50%, such as a reduction to less than 30%, such as a reduction to less than 200/o, for example a reduction to less than 10% of the binding of said histone H4 or fragment thereof with said TONSL, TONSL ARD or functional homologue thereof observed in the absence of said putative inhibitor. In one embodiment, the reduction is that there is no detectable binding between said histone H4 or fragment thereof and said TONSL, TONSL ARD or a functional homologue thereof.

High-throughput (HT) Biological Assays for Efficacy of Small Molecule Inhibitors in Killing Cancer Cells

Loss of TONSL leads to accumulation of replication associated DNA damage and sensitizes cells to camptothecin (CPT)-induced DNA damage (Duro et al. 2010 Mol Cell 40:619; O'Donnell et al. 2010 Mol Cel 40:619; O'Connell et al. 2010 Mol Cell 40:645; Piwko et al. EMBO 3 2010 29:4210). Further it is well established that loss of HR sensitizes cells to PARP inhibition. The prediction is thus that TONSL inhibition alone or in combination with CPT-like drugs or PARP inhibitors will be toxic to cancer cells.

Key Components:

-   -   a) Panel of cancer cell lines and primary cells     -   b) Campthotecin, PARP inhibitors, conventional chemotherapy         drugs targeting DNA replication

Rationale of HT Assays:

-   -   i. Assay the cell cytotoxicity of TONSL inhibitors by screening         a panel of cancer cell lines and primary cells.     -   ii. Assay the synergy of TONSL inhibitors and other cancer drugs         (conventional chemotherapy, CPT and derivatives, PARP         inhibitors) in killing cancer cells.

Typical Cell Toxicity Assay:

Grow cells in any HT format (e.g. 96 or 384 plates) and incubate with small molecule inhibitors in a dose and time dependent manner. Impact on cell proliferation and cell death may be analysed using using any suitable method available, e.g. colorimetric assays or HT microscopy.

In vivo Models for Drug Testing

Pre-clinical animal testing of TONSL inhibitors with the aim of starting clinical trials.

Small Molecules of the Invention

The small molecules of the present invention are useful for impairing homologous recombination. These could also involve both short linear and cyclic peptides and peptidomimetics, whose low molecular weights allows cell permeability.

By use of the structural data presented in the present application and the proof of concept assays above, the present inventors have identified pharmacophore targeted molecules, including small molecules, covalently linked small molecules, peptides or cyclic counterparts, interfering with the adjacent histone H4H18 and H4K20 binding pocket on the surface of the Ankyrin repeats of TONSL.

Structure Properties of the Small Molecule Inhibitor

The present invention relates to small molecule inhibitors, which target the conformational space of the TONSL ARD occupied by the histone H4 tail encompassing residues K12-R23 and act to disrupt the binding of the H4 tail K12-R23 with the TONSL ARD via direct competition or via allosteric disruption of the binding pocket.

In one embodiment, the present invention relates to a small molecule, which targets or interferes with the conformational space of the TONSL ARD occupied by the histone H4 tail encompassing residues K12-R23 and acting to prevent or disrupt the binding of the H4 tail K12-R23 with the TONSL ARD via direct competition or via allosteric disruption of the binding pocket.

As the skilled addressee would know, it's apparent that a small molecule that could bind to the His18-binding pocket or the Lys20-binding pocket or both pockets on TONSL ARD, would disrupt the interactions between H4 and TONSL.

In one embodiment, the present invention relates to a small molecule that targets the H4 tail spanning residues Lys12 to Arg23 through intermolecular hydrogen-bonding, electrostatic and/or van der Waals interactions.

In a more preferred embodiment, the present invention relates to a small molecule, wherein the molecule targets the intermolecular contacts spanning the Lys12-Gly13-Gly14-Ala15 segment of H4.

In another preferred embodiment the present invention relates to a small molecule, wherein the molecule targets the hydrophobic interactions between residues Gly13, Gly14 and Ala15 of H4 and residues Asn507, Cys508, Trp641, Tyr645 and Leu649 of ARD.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets the hydrogen bonds between the main-chain O of H4 Gly14 and NE1 of ARD Trp641, and between the main-chain N of H4 Ala15 and Oη of ARD Tyr645.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets the main-chain O of H4 Lys16 hydrogen bonds with the Nδ2of ARD Asn571.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets and/or associates with the conformational space of the TONSL ARD occupied by the side-chain of H4 Arg17, which stacks over the side-chains of ARD Tyr572 and Cys608, while its Nη1 atom forms two hydrogen bonds with main-chain O and Oδ1 of ARD Asn571. Thus, in one embodiment the invention relates to a compound, e.g. a TONSL inhibitor capable of associating with the side-chains of ARD Tyr572 and Cys608, and forming hydrogen bonds with the main-chain O and Oδ1 of ARD Asn571.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets and/or associates with the conformational space of the TONSL ARD occupied by the side-chain of H4 H18, which penetrates into a pocket lined by four strictly conserved residues (Trp563, Glu568, Asn571 and Asp604) and is positioned over His567 of ARD. Thus, in one embodiment the invention relates to a compound, e.g. a TONSL inhibitor capable of associating with Trp563, Glu568, Asn571, Asp604 and His567 of TONSL ARD.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets and/or associates with the conformational space of the TONSL ARD occupied by the side chain of H4 His18, which is stacked between Trp563 and Asn571 and forms hydrogen bonds to Glu568 and Asp604 of ARD. Thus, in one embodiment the invention relates to a compound, e.g. a TONSL inhibitor capable of associating with Trp563 and Asn571 and forming hydrogen bonds to Glu568 and Asp604 of TONSL ARD.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets and/or associates with the conformational space of the TONSL ARD occupied by the main-chain O of H4 Arg19 that forms a hydrogen bond with Nε1 of Trp563 and its side-chain forms contacts with Cys561 and Gly595 of ARD. Thus, in one embodiment the invention relates to a compound, e.g. a TONSL inhibitor capable of associating with the NE1 of Trp563 and with Cys561 and Gly595of TONSL ARD.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets and/or associates with the conformational space of the TONSL ARD occupied by the H4 Lys20 residue, which is bound within an acidic surface pocket on ARD adjacent to the H4 His18 binding pocket.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets and/or associates with the conformational space of the TONSL ARD occupied by side-chain of H4 Lys20, which interacts with the side-chain of Met528 and contacts the edge of Trp563 of ARD, while the main-chain atoms of H4 Lys20 packs against Cys561 of ARD. Thus, in one embodiment the invention relates to a compound, e.g. a TONSL inhibitor capable of associating with the side-chain of Met528 and Trp563 and Cys561 of TONSL ARD.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets and/or associates with the conformational space of the TONSL ARD occupied by the Nζ atom of H4 Lys20, which forms three strong hydrogen bonds (distance <3 Å) with the side-chains of strictly conserved residues Glu530, Asp559 and Glu568 of ARD, which surround H4 Lys20 within a regular triangle-like alignment. Thus, in one embodiment the invention relates to a compound, e.g. a TONSL inhibitor capable of forming hydrogen bonds with the side chains of Glu530, Asp559 and Glu568 of of TONSL ARD.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets the intermolecular contacts spanning the Val21-Leu22-Arg23 segment of H4, which include contacts between side-chains of H4 Val21 with Tyr560 and Cys561 of ARD, while H4 Leu22 interacts with Asp527 and Met528 of ARD.

In one embodiment, the present invention relates to a small molecule, wherein the molecule targets and/or associates with the conformational space of the TONSL ARD occupied by the main-chain N of H4 Arg23, which forms a hydrogen bond with the main-chain O of Asp527 of ARD, while the side-chain packs against the side-chain of Tyr560 of ARD. Thus, in one embodiment the invention relates to a compound, e.g. a TONSL inhibitor capable of forming hydrogen bond(s) with the main-chain O of Asp527 of ARD, while the binding the side-chain of Tyr560 of TONSL ARD.

In one embodiment, the present invention relates to a small molecule according to the present invention capable of blocking histone reader domains in a protein selected from the group consisting of TONSL, BARD1 and ANKRD11.

Such molecules are typically identified and validated by the SCHRODINGER package to identify and validate covalently linked small molecule drugs targeted to the adjacent histone H4H18 and H4K20 binding pockets on the surface of the Ankyrin repeats of TONSL.

Relationship with BARD1

The topology of TONSL ARD domain including the histone-binding surface is highly similar to the ARD of BARD1 (FIG. 23). BARD1 is an obligate binding partner of BRCA1 and is required for most cellular and tumour-suppressor functions of BRCA1. The key residues involved in histone H4 binding are conserved in ARD of BARD1. Furthermore, N571 residue of TONSL ARD that is essential for histone H4 binding (FIGS. 10, 12) and recruitment of TONSL to chromatin (FIG. 21) and sites of DNA damage (FIG. 22) and viability (Figure) corresponds to the BARD1 N4705 cancer mutation (FIG. 23). Therefore, small molecule inhibitors against TONSL ARD are predicted to target BARD1 ARD domain as well. Given that both TONSL and BARD1 function in the same DNA repair pathway, small molecule inhibitors designed based on our invention that would target both TONSL and BARD ARD would predictably inhibit HR more efficiently and show more potent anti-cancer activity as compared to inhibitors specific for TONSL ARD only.

General

It should be understood that any feature and/or aspect discussed above in connections with the compounds according to the invention apply by analogy to the crystal structures and/or methods described herein.

The atomic coordinate's variation of the crystal structures of the present invention such e.g. 1Å up to 3Å are used interchangeably.

The following sequence data, structures, figures and examples are provided below to illustrate the present invention. They are intended to be illustrative and are not to be construed as limiting in any way.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

View of the overall structure of the TONSL-ARD-MCM2 HBD-H3-H4 tetramer complex showing the relative positions of two TONSL ARDs, two MCM2 HBDs and an H3-H4 tetramer.

FIG. 2

TONSL ARD consists of four ANK repeats and uses its elongated concave surface to target the H4 tail spanning residues 12 to 23.

FIG. 3

Intermolecular interactions between TONSL ARD and the H4 tail (12-23).

FIG. 4

The electrostatic potential surface of ARD showing the acidic concave surface-binding site for the H4 tail.

FIG. 5

Highlight of the inter-molecular interactions of H4 Arg 17 with TONSL ARD.

FIG. 6

Highlight of the inter-molecular interactions of H4 His18 with TONSL ARD.

FIG. 7

Highlight of the inter-molecular interactions of H4 Lys20 with TONSL ARD.

FIG. 8

Molecular details of the interactions of TONSL ARD with H4 tail region residues 12-15.

FIG. 9

Molecular details of the interactions of TONSL ARD with H4 tail region residues 21-23.

FIG. 10

In vitro pull-down of recombinant histones H3-H4 with recombinant GST-TONSL ARD wild type or indicated mutants.

FIG. 11

Immunoprecipitation of HA-SNAP-H4 wild-type (WT) and indicated mutants transiently transfected into GFP-TONSL U-2-OS cells.

FIG. 12

Immunoprecipitation of GFP-TONSL WT or indicated mutants from transiently transfected HeLa S3 cells.

FIG. 13

ITC analysis of TONSL ARD binding to H4 tail peptides with the indicated modifications.

FIG. 14

In vitro pull-down of recombinant TONSL ARD with biotinylated H4 tail peptides.

FIG. 15

In vivo pull-down of GFP-TONSL from cell extracts with biotinylated H4 tail peptides

FIG. 16

In vitro pull-down of recombinant TONSL-MMS22L heterodimer with biotinylated recombinant mononucleosomes, unmodified or di-methylated at K20. * indicates an unspecific band. (bottom) TONSL binding quantified relative to histones. Unpaired t-test: *, P<0.05; mean of 6 independent experiments is shown; whiskers, outliers.

FIG. 17

Pull-down of GFP-TONSL from cell extracts with biotinylated H4 tail peptides

FIG. 18

Immunoprecipitation of GFP-TONSL from solubilized chromatin of GFP-TONSL U-2-OS cells

FIG. 19

Immunoprecipitation of GFP-TONSL from solubilized chromatin of GFP-TONSL U-2-OS cells

FIG. 20

High-content quantitative imaging of TONSL in pre-extracted U-2-OS fibroblasts. Plots show total TONSL and DAPI intensities in cells treated with control or TONSL siRNAs, confirming the specificity of TONSL antibody. Dots represent measured intensities of individual cell nuclei

FIG. 21

TONSL recruitment to chromatin requires recognition of H4K20me0. (a) U-2-OS cells conditional for GFP-TONSL WT or N571A were directly fixed or pre-extracted to remove soluble proteins. Scale bar, 20 μm. (b) (left)Analysis of GFP-TONSL WT and mutants by cellular fractionation. (right) The chromatin-bound fraction (C) was quantified by western blotting relative to total GFP-TONSL (error bar, SD; n≥3). S—soluble fraction, C—chromatin bound fraction. Staining with MemCode (ThermoFisher) was used to control the protein loading

FIG. 22

TONSL recruitment to DNA lesions requires recognition of H4K20me0. (top) U-2-OS cells conditional for GFP-TONSL WT or N571A were laser-irradiated and co-stained for 53BP1 and cycllin B to mark DNA damage and identify S/G2 cells, respectively. Full arrowheads, GFP-TONSL recruitment; empty arrowheads, no recruitment. Scale bar, 10 μm. (bottom) Quantification of GFP-TONSL cells showing recruitment to laser tracks (error bars, SD; n=3)

FIG. 23

Superposition of the structure of TONSL ARD and BARD1 ARD. The main residues involved in TONSL ARD interactions with the H4 tail are compared to the corresponding residues of BARD1 ARD. The two ARDs show highly similar topology and conservation of the histone-binding surface

FIG. 24

Co-localizationanalysis of chromatin-bound GFP-TONSL with MCM2 (A) and EdU (B) in inducible cell lines. Cells were either pulsed with EdU (left) or released into S phase in continuous presence of EdU (right). Co-localization analysed by deconvolution microscopy and measurement of Pearson coefficient in single cells (n>15, two independent experiments). Representative images,

FIG. 25

Chromatin-binding of GFP-TONSL WT and ARD mutants analysed across the cell cycle (representative of 2 experiments).

FIG. 26

Chromatin-binding of GFP-TONSL ARD WT and mutant analysed by high-content imaging of pre-extracted inducible U-2-OS cells treated with CPT (3 hours, 1μM CPT) as indicated (representative of 3 experiments)

FIG. 27

-   -   A) Co-immunoprecipitation analysis of TONSL-MMS22L with         Flag-HA-MCM2 WT or histone binding mutant of MMS22L(Y81A, Y90A)         isolated from solubilized chromatin from HUtreated cells (2         hours, 3mM) (representative of 2 experiments).     -   B) Analysis of GFPTONSL ARD WT and mutant recruitment to         CPT-challenged replication forks by NCC as illustrated (CPT,         1μM). (−), no biotin-dUTP.

FIGS. 28 and 29

Colony formation upon GFP-TONSL expression induced by tetracycline in TONSL depleted (FIG. 28) and control cells (FIG. 29). Cells were siRNA treated and induced to express siRNA-resistant GFP-TONSL ARD WT and mutant by tetracycline (tet) and treated with CPT (24 hours, 50nM) as indicated. Rescue efficiency determined by colony forming efficiency of TONSL depleted cells (A). Dominant negative effect of TONSL ARD mutant determined by colony forming efficiency of cells treated with control siRNA (B). Data points represent two different cell concentrations in technical triplicate from two or four (28, left panel) independent biological replicates.

FIG. 30

A) Colony formation upon GFP-TONSL expression induced by tetracycline in TONSL depleted and control cells as shown in FIG. 4f but including additional ARD mutants. Two cell concentrations in technical triplicate from two (E568A, D559A) or four (WT, N571A) biological replicates are shown. B) Representation of the complementation analysis from FIG. 4f in a single panel including both CPT treated and untreated cells.

This illustrates that the toxicity of the TONSL ARD mutant is comparable to CPT treatment of cells expressing WT TONSL.

FIG. 31

Cells were treated as in FIG. 29 and analysed for cell cycle distribution using EdU and Dapi (left) and 53BP1 foci (rigth). Error bars, mean with S.D (four (left) or five (right) biological replicates in technical triplicates).

FIG. 32

Chromatin-binding of MMS22L. The chromatin-bound fraction was analyzed by western blotting (B) and quantified relative to total MMS22L (A) (untreated: error bar, SD, n=3; CPT: error bar, mean with range, n=2).

FIG. 33

Pull-down of recombinant histones H3-H4 and MCM histone binding domain (HBD) with GST-TONSL ARD WT or indicated mutants.

FIG. 34

Circular dichroism (CD) analysis of TONSL ARD WT and the indicated ARD mutants.

The indicated ARD point mutations do not destabilize the overall structure of the ARD.

FIG. 35

ITC analysis of TONSL acidic stretch and ARD (aa. 450-692) with H3K9me1 (aa. 1-21) and H4 (aa. 9-25).

FIG. 36

Analysis of TONSL chromatin-binding in MOF-depleted cells. Chromatin-bound TONSL was quantified by high content imaging of pre-extracted cells stained for endogenous TONSL. Mean TONSL intensity is shown. A.U., arbitrary units. Knock-down efficiency and expected effect on histone modification were confirmed by western blotting (representative of 2 experiments).

FIG. 37

H4K20 methylation levels measured by mass spectrometry in synchronized TIG3 cells. Cell were treated with HU (3 mM) or CPT (1 μM) from 3-6 hours after release or left untreated (6 hours) (error bars, range; n=2).

EXAMPLES Example 1 The Crystal Structure

Protein production: All proteins described in the Examples herein, unless otherwise indicated, were expressed in BL21(DE3)-RIL cell strain (Stratagene). The GST-tagged TONSL ARD and its mutants including E530A, D559A, W563A, E568A, N571A and D604A were cloned into pGEX-6P-1 vector (GE Healthcare). The expressed proteins were first purified using Glutathione Sepharose 4B, then further purified by gel-filtration step. In some case, the GST-tag was removed with 3C protease before gel-filtration step. For purification of GST-H3 tail and GST-H4 tail proteins, the human histones H3 fragment 1-59 and H4 fragment 1-31 were cloned into pGEX-6P-1 vector respectively. The proteins were expressed and purified in the same way.

For production of recombinant full-length TONSL-MMS22L heterodimer, the sequence coding for full-length MMS22L was fused with a MBP tag at the 5′ end and 10×His tag at the 3′ end. The sequence coding for full-length TONSL was fused with GST tag at the 5′ end. Both MMS22L and TONSL constructs were cloned into a pFastBacl vector. The complex was expressed in Sf9 cells by co-infection with both recombinant baculoviruses according to manufacturer's recommendation (Invitrogen). The proteins were extracted from Sf9 cells and purified similarly as described previously for Sgs1. Briefly, the complex was purified on amylose resin, and MBP and GST tags were subsequently cleaved with PreScission protease. The heterodimer was then further purified using a Ni-NTA affinity resin. Washes were performed with 300 mM NaCI buffer.

For crystallization: The human TONSL Ankyrin Repeat Domain (ARD, residues 512-692) and MCM2 Histone-binding Domain (HBD, residues 61-130) were covalently linked through a four-Glycine linker (G₄ linker) into a single expression cassette (SEQ ID NO: 15).

The MCM2 HBD-G₄-TONSL ARD expression cassette was cloned into a modified RSFDuet-1 vector (Novagen), with an N-terminal His6-SUMO tag. The resulting plasmid was coexpressed with a pETDuet plasmid harboring human histone genes H3.3(57-135) and H4(1-102) in BL21(DE3)-RIL cell strain (Stratagene).

The E. coli was cultured at 37° C. using LB media with 50 μg/ml Kanamycin, 100 μg/ml Ampicillin and 34 μg/ml Chloramphenicol.

When the E. coil reached cell density of OD₆₀₀˜1.0, 0.5 mM IPTG was added into the LB media which was further incubated at 20° C. overnight.

The expressed protein complex was first purified on HisTrap HP column (GE Healthcare). After removing the His₆-SUMO tag by using Ulp1 (SUMO protease), the protein complex was further purified on HiLoad 16/600 Superdex 200 column (GE Healthcare) in the buffer of 20 mM Tris pH 7.5 and 500 mM NaCl.

The purified G4 linker complex, MCM2 HBD-G₄-TONSL ARD cassette-H3.3(57-135)-H4(1-102) complex (herein designated as TONSL ARD-MCM2 HBD-H3-H4 tetramer complex) with a concentration of 23 mg ml⁻¹ in the buffer of 20 mM Tris pH 7.5 and 1 M NaCl, was crystallized in the condition of 100 mM MES pH 5.6-6.6, 5-10% isopropanol using setting-drop vapor-diffusion method at 20° C. All the crystals were soaked in a cryoprotectant made from the mother liquor supplemented with 25% glycerol before flash freezing in liquid nitrogen.

The data set for the TONSL ARD-MCM2 HBD-H3-H4 tetramer complex was collected at 0.979 Å on 24-ID-C/E NE-CAT (Advanced Photo Source, Argonne National Laboratory). The data was processed using the HKL 2000 program. The initial structure for the complex was solved by molecular replacement in PHASER with our previous structure of the MCM2 HBD-H3-H4 tetramer complex as a search model and manually refined and built using Coot. The final structure of this complex was refined to 2.43 Å resolution using PHENIX. Table 1 summarizes the statistics for data collection and structural refinement

TABLE 1 Data collection and refinement statistics TONSL ARD-MCM2 HBD-H3-H4 Tetramer Complex Data collection Space group P3 2 1 Cell dimensions a, b, c (Å) 139.5, 139.5, 72.9 α, β, γ (°) 90, 90, 120 Resolution (Å) 50-2.43 (2.95-2.43)^(a) R_(pim) (%)  3.8 (46.8) I/σI 23.1 (1.8)  Completeness (%) 99.8 (99.7) Redundancy 5.5 (5.5) Refinement Molecules per 1 asymmetric unit No. reflections 171,308/31,146 (total/unique) R_(work/)R_(free) (%) 20.1/24.6 No. atoms Protein 2,908 MES 12 Glycerol 12 Water 87 B-factors Protein 81.8 MES 108.5 Glycerol 92.6 Water 59.8 R.m.s deviations Bond lengths (Å) 0.009 Bond angles (°) 1.316 Ramachandran plot^(b) Favored (%) 95.9 Allowed (%) 4.1 ^(a)Highest resolution shell is shown in parenthesis. ^(b)Calculated using MolProbity in PHENIX.

The structural data obtained are described below in Annex 1.

One molecule of each protein MCM2 HBD-TONSL ARD cassette, H3 and H4 is present in the asymmetric unit. The crystallographic symmetric operation reconstitutes a tetramer of H3-H4, thus resulting in formation of an intact complex with two copies of MCM2 HBD-TONSL ARD cassette in complex with an H3-H4 tetramer (named TONSL ARD-MCM2 HBD-H3-H4 tetramer complex), which is consistent with our previous finding that MCM2 HBD binds and stabilizes an H3-H4 tetramer under physiological conditions. The TONSL ARD-MCM2 HBD-H3-H4 tetramer complex is also highly similar to our previous structure of the MCM2 HBD-H3-H4 complex, with a pair of MCM2 HBDs wrapping around the lateral surface of the H3-H4 tetramer, while the two TONSL ARDs interact with each of the H4 tails. The TONSL ARD forms no intermolecular interactions with the MCM2 HBD, consistent with the H3-H4 tetramer bridging the interaction between TONSL and MCM2 in cells. The TONSL ARD forms extensive contacts with a segment of the H4 tail (residues 12-23), but shows only minimal contacts with the core of the H3-H4 tetramer. The TONSL ARD could be modeled to bind the H4 tail in the context of the nucleosome without steric clashes and a conserved positive patch may interact with the nucleosomal DNA, suggesting that the extensive interactions between TONSL ARD and the H4 tail could describe TONSL binding to both soluble non-nucleosomal histones H3-H4 together with MCM2 and also to H3-H4 in nucleosomes.

The Ankyrin (ANK) repeat fold contains two antiparallel helices named the inner and outer helix respectively, followed by a hairpin loop named the finger. The TONSL ARD consists of four ANK repeats, three of which adopt the canonical ANK repeat fold (ANK1-3), while the remaining one is an atypical and capping repeat (ANK4). Besides the internal fingers 1-3, the TONSL ARD contains an extra loop preceding ANK1, designated finger 0. The TONSL ARD uses its elongated concave surface composed of inner helices (aA1, aB1, aC1 and aD1) and fingers 0-4 to form extensive intermolecular contacts with the extended β-strand like conformation of the H4 tail. It is notable that 15 out of 18 residues that constitute the H4 tail-binding surface of TONSL ARD are highly conserved. The TONSL ARD targets the H4 tail spanning residues Lys12 to Arg23, primarily through intermolecular hydrogen-bonding, electrostatic and van der Waals interactions.

Example 2 Determination of TONSL ARD Binding to Histone H4 Peptides by Isothermal Titration Calorimetry (ITC)

All the ITC titrations were performed on a Microcal ITC 200 calorimeter at 25° C. The peptides of histone H4 (residues 9-25) and its modified peptides K16ac (with acetylation on Lys16), H18W (with His18 mutated to Trp18), H4K20me1 (mono-methylation on Lys20) and H4K20me2 (di-methylation on Lys20), and peptide of histone H3(1-19)K9me1 (mono-methylation on Lys9) were all synthesized at Tufts University Core Facility. The exothermic heat of the reaction was measured by 17 sequential 2.2 μl injections of the peptides (1.41 mM in buffer 20 mM Tris pH 7.5 and 0.5 M NaCl) into 200 μl of the TONSL ARD solution (145 μM in the same buffer), spaced at intervals of 150 s. The data were processed with Microcal Origin software and the curves were fit to a single size binding model.

Example 3 Assaying Binding of Recombinant TONSL ARD to Histone H4 Peptides In Vitro

Purified recombinant TONSL ARD (residues 512-692) was stored at 400 μM concentration in 1 M NaCl, 20 mM Tris HCl pH 7.5 at −80° C. For each pull-down, 400 pmol of the ARD stock (1 pi, 400 μM) was diluted with 99 μl of binding buffer (150 mM NaCl, 50 mM Tris HCl pH 7.5, 5% Glycerol, 0.25% NP-40, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 1 mM Leupeptin, 1 mM Pepstatin). ARD input material was scaled to the number of pull-downs performed. For each pull-down, a histone H4 peptide OPT Peptide Technologies GmbH) spanning residues 14-33 (2.5 μl, 250 μM) with a C-terminal biotinoyl-lysine residue or biotin (2.5 μl, 400 μM) was added to 1.1 ml of binding buffer in addition to 100 μl of the ARD input material and the mixture incubated overnight rotating at 4° C. The next day 25 pl of MyOne Streptavidin C1 beads (Life Technologies) was washed in binding buffer (3×500 μl) for each pull-down removing the final wash from the beads. The ARD/peptide or ARD/biotin mixture was added to an aliquot of pre-washed MyOne Streptavidin C1 beads and incubated with rotation at 4° C. for 3 hours. Finally the beads were washed (2×300 pl and 1 ×200 μl of 300 mM NaCl, 50 mM Tris HCI pH 7.5, 5% Glycerol, 0.25% NP-40, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 1 mM Leupeptin, 1 mM Pepstatin) and pulldown material visualized by Coomassie staining after SDS PAGE separation of proteins on a NuPAGE 4-12% gel.

Example 4 Measuring GFA-TONSL Binding to Histone H4K2Ome0 peptides in Cell Extracts

For detergent-soluble extracts (NP40/NaCl), U-2-OS cells expressing GFP-TONSL WT or ARD mutants were washed with cold PBS, scraped and incubated for 15 min on ice in HS buffer supplemented with trichostatin A (TSA) and protease and phosphatase inhibitors (5 mM sodium fluoride, 10 mM μ-glycerolphosphate, 0.2 mM sodium vanadate, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 0.1 mM PMSF, Sigma). After centrifugation at 16.000 g for 15 min at 4° C., the supernatant was collected. For pull-downs from cell extracts, MyOne T1 beads were incubated O/N with 1 μg of biotinylated histone peptides in High Salt (HS; 300mM NaCl, 0.5% NP40, Tris HCl, EDTA, 5% glycerol) buffer and subsequently washed 2 times with PBS. 1 mg of NP40/NaCl extract from GFP-TONSL U-2-OS cells was added to the beads and incubated for 2 hrs rotating at 4° C. The beads were then washed 5 times with HS buffer, 2 min rotating at 4° C. After washing, the beads were resuspended in 1XLSB and boiled for 10 min. The eluted proteins were loaded on a 4-12% Bis-Tris NuPage gel (LifeTechnologies). Proteins were then transferred to a 0.2 pm nitrocellulose membrane by O/N wet transfer at 20V and detected by western blotting.

Example 5 Quantifying Chromatin-Bound TONSL in Human Cells by HT Microscopy

U-2-OS and TIG3 cells were grown on 6-well plates (1×10⁵ cells seeded/well 1 day prior to analysis). To analyse only chromatin-bound TONSL, soluble proteins were removed (pre-extracted) by 5 min incubation on ice with CSK buffer 0.5% Triton (CSK buffer: 10 mM PIPES pH 7, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 plus protease and phosphatase inhibitors 1 mM DTT, 10 ug/ml leupeptin, 10 ug/ml pepstatin, 0.1 mM PMSF, 0.2 mM sodium vanadate, 5 mM sodium fluoride, 10 mM beta-glycerolphosphate). Cells were then rinsed with CSK and PBS before fixation in 4% formaldehyde for 10 min and staining with TONSL antibody.

For detection, cells were blocked with PBS containing 5% BSA and 0.1% Tween20 for 1 h, and incubated with TONSL antibody (Sigma, ref. nr. HPA0244679) overnight at 4° C. (1:400 in blocking buffer). After washing 3 times with PBS containing 5% BSA and 0.1% Tween 20, anti-rabbit-Alexa488 (LifeTechnologies 1:1000 in blocking buffer) was applied and let to incubate for 30 min. Cells were washed 3 times and DNA was counterstained with DAPI (Sigma). Images were acquired on ScanR high-content imaging system (Olympus) and analysed using ScanR software. Relative fluorescence intensity TONSL was quantified relative to cells depleted for TONSL using the specific siRNAs (O'Donnell et al. 2010 Mol Cel 40:619, synthetized by Sigma) for 30 h at 100 nM concentration.

Example 6

Given that TONSL-MMS22L binds histones in a pre-deposition complex with ASF1 and MCM2, TONSL-MMS22L could be loaded onto replicating DNA together with new histones. It is shown herein that in nascent chromatin, new histones were exclusively unmethylated at H4K20 (98% H4K20me0), while old recycled histones were almost fully methylated at H4K20 (mel, 7%; me2, 88%; me3, 2%). New histones became methylated in late G2/M, rendering G1 chromatin devoid of H4K20me0. This identifies H4K20me0 on new histones as a signature of post-replicative chromatin, implying that TONSL-MMS22L can bind H4 tails on new histones at replication forks and sister chromatids until late G2/M. Confirming this prediction, TONSL accumulated on chromatin in S phase, remained chromatin-bound in a population of G2 cells and was excluded from chromatin in G1 (FIG. 20). To discriminate pre- and post-replicative chromatin, we labeled replicating DNA with EdU (pulse to mark ongoing replication, continuous labeling to identify post-replicative chromatin) and marked pre-replicative chromatin with MCM2, and analyzed co-localization with TONSL. TONSL staining was mutually exclusive with MCM2 (FIG. 24A), but co-localized with EdU pulse labeling in very early S phase and with replicated DNA (continuous EdU labeling) throughout S phase (FIG. 24B). TONSL was present at sites of ongoing DNA replication throughout S phase, but the degree of co-localization declined in mid/late S (FIG. 24B, left panel), consistent with TONSL binding to post-replicative chromatin also after fork passage (FIG. 24B, right panel). Mutation of the TONSL ARD abrogated recruitment of TONSL to chromatin, including DNA replication sites (FIG. 25,). Together, this demonstrates that TONSL is recruited to replication forks and post-replicative chromatin via ARD recognition of H4K20me0 on new histones. TONSL ARD recognition of the H4 tail is required for binding to post-replicative chromatin and recruitment of damaged forks and DNA lesions.

Mutation of TONSL ARD also abrogated chromatin binding (FIG. 26) and recruitment to replication forks in the presence of replication poisons like camptothecin (CPT) and hydroxyurea (HU) (FIG. 27A and B). Furthermore, ARD mutation prevented accumulation of TONSL at site-specific DSBs (FIG. 28) and microlaser-generated DNA damage. Co-staining with cell cycle markers confirmed that TONSL is recruited to DNA repair sites only in S and G2 cells as expected. H4K20me0 binding is required for TONSL accumulation at damaged forks and DNA lesions in post-replicative chromatin. However, this was not due to increased H4K20me0, suggesting that unmasking of H4 tails upon chromatin decompaction and/or interaction with repair factors contribute to TONSL-MMS22L accumulation at repair sites. Consistent with the latter, MMS22L interaction with Rad51 can stabilize the complex at challenged forks (P. Cejka and M. Peter, personal communication). Our data argue that this is subsequent to H4K20 binding (FIGS. 26 to 28). In complementation analysis, TONSL WT partially rescued viability of TONSL depleted cells in the presence and absence of CPT (FIG. 28, FIG. 30A and B), whereas TONSL ARD mutants were highly toxic (FIG. 28, FIG. 30A and B). In control cells, TONSL ARD mutants also reduced viability and caused G2/M arrest accompanied by replication-associated DNA damage (FIG. 29 and FIG. 31). Further, the TONSL ARD mutant titrated MMS22L away from chromatin, explaining the dominant negative phenotype that mimics TONSL-MMS22L depletion (FIG. 32A and B). Collectively, this indicates that recognition of H4K20me0 is central to TONSL-MMS22L function in safeguarding genome stability.

The cell viability was assayed by clonogenic assay (see Example 8 herein below for details of the assay) using inducible U205 cells that express siRNA resistant GFP-TONSL WT or ARD mutant (D559A, E568A, N571A), in the presence or absence of CPT. Aforementioned results shows that GFP-TONSL WT can partially complement the growth defect and CPT sensitivity of TONSL depleted cells, while expression of GFP-TONSL ARD mutants further impairs cell viability and survival to CPT. Also, it show that expression of GFP-TONSL ARD mutants in control cells has a dominant negative effect on cell growth.

Accumulation of the genome instability marker 53BP1 and cell cycle arrest in G2/M in cells expressing GFP-TONSL ARD mutants was determined. Inducible U2OS cells were induced to express resistant GFP-TONSL WT or ARD mutant (D559A, E568A, N571A) for 24 hours and then fixed and stained for 53BP1 and EdU after an additional 24 hours. Images were acquired by high-throughput microscopy. This shows that expression of the TONSL ARD mutants phenocopies TONSL/MMS22L depletion. Accordingly, these mutants are useful for testing the effect of inhibitors of TONSL in various disease models. The demonstrated titration of MMS22L away from chromatin in cells expressing GFP-TONSL ARD mutants, both in the absence and presence of CPT may explain why expression of TONSL ARD phenocopies TONSL/MMS22L depletion.

It is revealed that post-replicative chromatin has a distinct histone modification signature, read by the TONSL-MMS22L effector protein. This opens a new avenue to understand how DNA repair and other chromosomal transactions can be directly linked to the replication state of a genomic locus. Intriguingly, it is the new histones that make post-replicative chromatin distinct. It is presented that TONSL-MMS22L is delivered to nascent chromatin with new histones via the pre-deposition complex with MCM2 and ASF1. TONSL may thus have a dual function as a histone chaperone and histone reader. The structural work proposes that TONSL acts in a histone chaperone-like capacity by sequestering the H4 tail to prevent spurious contacts with DNA during H3-H4 deposition. Further, TONSL ARD may counteract chromatin compaction by preventing association of the H4 tail with the H2A-H2B acidic patch on neighboring nucleosomes. Thus, TONSL changes our perception of a histone chaperone by binding both soluble and nucleosomal histones. In its function as a histone reader, TONSL localizes MMS22L to post-replicative chromatin via H4K20me0 and allows TONSL-MMS22L to accumulate at damaged forks and DNA lesions. We envision that H4K20me0 works as an affinity trap, making TONSL-MMS22L readily available to support Rad51 loading during HR. This provides a new angle to understand the role of H4K20 in DNA repair, complementing the well-described role of H4K20me1/2 in recruiting 53BP1 to promote NHEJ in competition with BRCA1-BARD1. In post-replicative chromatin, H4K20me1/2 on old histones will support 53BP1 recruitment. Whether H4K20me0 on new histones also influences DNA repair pathway choice will be of interest to future investigations. It is notable that the structure of the TONSL ARD, including the histone-binding surface, is highly similar to the ARD of BARD1, required for BRCA1 tumor suppressor function and HR. Multiple mutations in the TONSL ARD are reported in cancer (C608G, COSM4879909; P557S, COSM4565032; E597K, COSM3382163) and the N571 residue, key to H4 binding, corresponds to the BARD1 N470S cancer mutation. This underscores that the tumor suppressor function of H4K20me0 recognition and the possibilities it brings for targeted cancer therapy should be explored in the future.

Circular dichroism analysis of TONSL ARD WT or mutants stability (FIG. 34), show that the indicated ARD point mutations do not destabilize the overall structure of the ARD. ITC analysis of TONSL acidic stretch and ARD (450-692) with H4 tail (9-25) and H3K9me1 (1-21) peptides (FIG. 35) show that TONSL (450-692) does not bind to H3K9me1, but recognizes H4 peptides.

The methods used for immunofluorescence, microscopy and laser microirradiation are described below in Example 7.

Example 7

Immunofluorescence, Microscopy and Laser Microirradiation

U-2-OS, HeLa 53, and TIG-3 cells were grown in DMEM (Gibco) containing 10% FBS (Hyclone) and 1% penicillin/streptomycin and drugs for selection. The construct for siRNA resistant GFP-TONSL was described (O'Donnell et al., Mol Cell 40, 619-631 (2010)) and ARD mutation were introduced in this construct by site-directed mutagenesis. Cells inducible for GFP-TONSL WT and ARD mutants were generated in Flp-In T-Rex U-2-OS cells (Invitrogen) by transfection of pcDNA5/FRT/TO-GFP-TONSL plasmids with Lipofectamine 2000, according to the manufacturer's protocol, and selection with hygromycin (200 μg/ml). All cell lines were authenticated by western blotting and immunofluorescence. Expression of GFP-TONSL was induced by addition of 1 μg/ml of tetracycline for 24 hours. U-2-OS and TIG3 cells were synchronized by a single thymidine block (2mM) and released into S phase in the presence of 24 □M dCTP. For transient expression of GFP-TONSL, expression plasmids were introduced by transfection with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol and cells harvested 24 hours after transfection. siRNA transfection was performed with RNAiMax reagent (Invitrogen) according to the manufacturer's protocol.

siRNA sequences: siSET8#1: 5′-GUACGGAGCGCCAUGAAGU-3′; siSET8#2: 5′-ACUUCAUGGCGCUCCGUACUU-3′; siM0F#1: 5′-GUGAUCCAGUCUCGAGUGA-3′; siMOF#2: 5′-GUGAUCCAGUCUCGAGUGA-3′; siTONSL: 5′-GAGCUGGACUUAAGCAUGA-3′. All cell lines used in this study tested negative for mycoplasma contamination. U-2-OS cells conditional for GFP-TONSL were grown on glass coverslips and either directly fixed in 4% paraformaldehyde (PAF) for 10 mins or washed in CSK, pre-extracted 5 min with CSK/0.5% Triton X-100 and rinsed with CSK and PBS before fixation in 4% PAF for 10 mins. Coverslips were mounted on glass slides with Mowiol mounting medium (Sigma Aldrich) containing DAPI. Fluorescence images were collected on a DeltaVision system with a 40× or 60× oil immersion objective. For deconvolution microscopy, z-stacks were acquired (step of 0.2 □m), deconvolved and analyzed by SoftWoRX 5.0.0. Pearson coefficient correlation analysis was performed on single cells using SoftWoRX 5.0.0. Brightness and contrast were adjusted using Adobe Photoshop CS6. For high content quantitative analysis, fluorescence images were acquired using an Olympus ScanR high-content microscope and processed on the ScanR analysis software. More than 5000 cells per sample were analyzed. Graphs were generated with TIBCO Spotfire software. For microirradiation experiments, cells grown on glass coverslips were fixed in 4% formaldehyde for 15 min, permeabilized with PBS containing 0.2% Triton X-100 for 5 min and incubated with primary antibodies diluted in DMEM for 1 hour at room temperature. Following staining with secondary antibodies (Alexa Fluor 488, 568 and 647; Life Technologies) for 30 min, coverslips were mounted on glass slides in Vectashield mounting medium (Vector Laboratories) containing the nuclear stain DAPI. For detection of nucleotide incorporation during DNA replication, an EdU-Plus labeling kit (Life Technologies) was used according to the manufacturer's instructions. Confocal images were acquired on an LSM-780 (Carl Zeiss) mounted on a Zeiss-AxioObserver Z1 equipped with a Plan-Neofluar 40×/1.3 oil immersion objective. Image acquisition and analysis was carried out with LSM-ZEN software. Laser microirradiation of cells was performed essentially as described.

Example 8

Clonogenic Assay

U-2-OS inducible for GFP-TONSL ARD WT and mutant were transfected with siRNA, trypsinized 24 hours later and seeded in technical triplicates of 1000 or 3000 cells in the presence or absence of tetracycline. After 24 hours, the cells were washed and left in fresh medium for 12-15 days before fixation and staining with MeOH/Crystal Violet. Colony formation efficiency was determined by manual colony counting or quantification of Crystal Violet staining by Image] software and normalized to non-induced control.

Example 9

Nascent Chromatin Capture (NCC)

The NCC protocol from (Alabert et al., Nat Cell Bial 16, 281-293 (2014)) was adjusted for adherent U-2-OS cells. CPT (1μM) was added 5 minutes prior to b-dUTP labelling and was included in all steps until fixation. Cells were incubated for 5 minutes in a hypotonic buffer (50 mM KCl, 10 mM Hepes) containing biotin-dUTP and resuspended into fresh cell culture medium for an additional 15 minutes. Cells were fixed 15 minutes in 1% formaldehyde, rinsed twice in PBS and collected by scraping in cold room. Nuclei were mechanically isolated in sucrose buffer (0.3 M sucrose, 10 mM HEPES-NaOH at pH 7.9, 1% Triton X-100 and 2mM MgOAc). Chromatin was solubilized by 28 cycles 30 sec ON, 90 sec OFF in sonication buffer (10 mM HEPES-NaOH at pH 7.9, 100 mM NaCl, 2 mM EDTA at pH 8, 1 mM EGTA at pH 8, 0.2% SDS, 0.1% sodium sarkosyl and 1 mM phenylmethylsulphonylfluoride) using a Bioruptor at 4° C. Solubilized chromatin was pre-cleared using streptavidin-coated magnetic beads (MyC1 Streptavidin beads) pre-incubated with biotin. b-dUTP labelled chromatin was next purified over night at 4° C. using streptavidin-coated magnetic beads. Beads were washed 5 times for 2 minutes in wash buffer (10 mM HEPES-NaOH pH 7.9; 200 mM NaCl; 2 mM EDTA pH 8; 1 mM EGTA pH 8; 0.1% SDS; 1 mM PMSF). Total chromatin (input) and isolated nascent chromatin were boiled 40 min on beads in LSB 1× (50 mM Tris-HCl pH 6.8, 100 mM DTT, 2% SDS, 8% Glycerol, Bromophenol blue) and separated by SDS-PAGE for western blotting. An alternative method which may be used in place of NCC is iPOND (Sirbu et al., Nat Protoc 3, 594-605 (2012)).

Example 10

Native MS Analysis of Protein-Peptide Complexes

Various peptides were obtained including the peptides described in Examples 11, 12, 13, 14 and 15. If the peptide was in the form of a trifluoroacetate salt (TFA salt), it was desalted on a polymeric weak cation exchange sorbent (Strata X-CW, Phenomenex, Sartrouville, France): Dry peptide (1-3 pmol) were solubilized in 200 μL pure water (Sigma-Aldrich, Lyon, France). The pH of the solution was adjusted to 7-7.5 by addition of 1 mM aqueous ammonium hydroxide solution (solution freshly prepared from 20% concentrated ammonium hydroxide). The exchange column was conditioned with 1 mL methanol (Sigma-Aldrich, Lyon, France) followed by 1 mL water. The solution was injected and the column was washed twice with 500 μL pure water. Finally the peptide was eluted by gravity with 2 times 500 μL of a 20:80:5 acetonitrile/water/formic acid mixture. The elution fraction containing the expected peptide was identified by mass spectrometry. The TFA-free solution was freeze-dried to get a white solid. The solid was dissolved in 300 μL pure water and the solution was freeze-dried for a second time.

A stock solution of the peptide was prepared by adding 300 μL of pure water to the resulting solid and the concentration of the solution was measured by UV at a wavelength of 205 nm. The extinction coefficient for the corresponding peptide sequence was calculated using the Nick Anthis online protein parameter calculator script (http://nickanthis.com/tools/a205.html). The absence of TFA adducts was confirmed by mass spectrometry with infusion of the protein-peptide mixture at low acceleration voltage (Vc 25 V) in native conditions.

Purified TONSL ARD (i.e. amino acid 512-692 of TONSL of SEQ ID NO:16) was buffer exchanged against 500 mM NH₄OAc pH 7.5 using a NAP 5 column (NAP™-5, GE Healthcare). Bradford protein quantitation was performed on buffer exchanged protein. Protein was diluted in water to a final concentration of 10 μM and kept on ice until native MS analyses were performed.

The mass spectrometry (MS) analysis were carried out on an electrospray time-of-flight mass spectrometer (LCT, Waters, Manchester, UK) equipped with an automated chip-based nanoESl device (Triversa Nanomate, Advion Biosciences, Ithaca, N.Y.). External calibration was done in the positive ion mode over the mass range m/z 200-6000 using the multiply charged ions produced by 0.4 μM horse heart myoglobin solution diluted in water/acetonitrile 50/50 mixture acidified with 0.5% (v/v) formic acid. Homogeneity and purity of TONSL-ARD was first checked under denaturing conditions by diluting the protein to 1 μM in 50/50 water/acetonitrile mixture acidified with 0.5% (v/v) formic acid. Mass measurement revealed mainly the presence of a species with a molecular weight of 20747.18±0.34 Da. Characterization of peptide binding to TONSL-ARD under native conditions was performed in 50 mM NH₄OAc pH 7.5 keeping a constant 5% amount of EtOH (v/v). The protein concentration was set to 10 μM and different peptide concentrations ranging from 1 to 5 molar equivalents were added. Incubations were performed at room temperature for 15 min. Mass spectra were recorded using reduced cone voltage (Vc=50 V) and elevated interface pressure (Pi=5 mbar) which correspond to fine-tuned instrumental settings providing sufficient ion desolvation while preserving the integrity of weak non-covalent complexes in the gas phase. Micromass MassLynx 4.1 was used for data acquisition and processing. Native MS analysis of protein-peptide complexes were attempted by incubation of 10 μM TONSL-ARD with several peptide concentrations (10, 20, 30, 40, 50 μM).

For data processing under native conditions, protein-peptide complex abundance (% PL) was estimated from the peak heights of 9⁺ and 8⁺ charge states of free and bound TONSL-ARD, assuming that compound binding does not alter protein response factor. The proportion of 1:1 stoichiometry complex is calculated according to the following equation:

${\% \mspace{14mu} P\; 1L\; 1} = \frac{I_{P\; 1L\; 1}}{I_{P\; 1L_{1\;}} + I_{P}}$

To estimate the binding affinities (Kd) of peptides for TONSL-ARD, a mixture of protein/peptide was analyzed at a constant peptide to protein ratio with decreasing protein concentration until the lowest detection limit was reached. For data processing under native conditions, protein-peptide complex abundance (% PL) was estimated from the peak heights of 9⁺ and 8⁺ charge states of free and bound TONSL-ARD. This protein proportion is used to calculate the bound protein, the free protein and the free peptide concentrations. The Kd is calculated by using the following equation for each protein/peptide concentration. The final Kd for each peptide is the average of the Kd at different protein/peptide ratio.

${Kd} = \frac{\left\lbrack {P\mspace{14mu} {free}} \right\rbrack \left\lbrack {L\mspace{14mu} {free}} \right\rbrack}{\lbrack{PL}\rbrack}$

Test Compounds

Example 11

Leu-Gly-Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-Leu-Arg-Asp-Asn-Ile-NH₂ (Histone H4 peptide)

Purchased from JPT Peptide Technologies GmbH, Berlin, Germany

The following peptides were obtained from Schafer-N Aps, Copenhagen, Denmark and were prepared by using Fmoc-chemistry on chlorotrityl resins, a method known to those skilled in the art.

Example 12

Ala-Lys-Arg-His-Arg-Lys-Val-Leu-Arg-NH₂

Example 13

Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-Leu-Arg-NH₂

Example 14

Lys-Gly-Gly-Ala-Lys-Arg-His-Ala-Lys-Val-Leu-Arg-NH₂

Example 15

Lys-Gly-Gly-Ala-Ala-Arg-His-Arg-Lys-Val-Leu-Arg-NH₂

Using the assays described in Examples 10, the following values were obtained.

Example % P1L1 Apparent K_(d) (μM) Titration K_(d) (μM) 11 0.077 12 62.6 2.2 1.95 13 89.9 0.1 0.31 14 73.3 1.0 0.96 15 81.4 0.4 not determined* *A Kd value could not be measured by titration

Example 16

Structure Based Design of Small Molecule and Peptide Inhibitors:

Using the structural information of the present invention, we conducted a large scale virtual screening of the collective repertoire of world vide vendor chemical libraries of available screening compounds (small molecules and peptides) directly related to drug discovery applications of ligands, which can be developed into potent and efficacious TONSL inhibitors. The screened (in silica) chemical libraries and the virtual screening protocol is described below.

Chemical Vendor Libraries:

The ‘In Stock’ subset of the ZINC database containing 12,782,590 biologically relevant screening molecules, that are stripped for counter ions and assigned tautomers, protonation states, charges and 3D conformations, was obtained from htt;//zinc. docking.org and stored in SDF files.

Preparation of the TONSL Structure for Docking:

a. The histone H4 tail (K12-D24) and crystallographic water molecules was removed from the TONSL ARD complex structure (see Example 1).

b. All hydrogen atoms were built and optimized using ICM (Molsoft L.L.C., San Diego, Calif., USA)

c. A box of interactions grids (20×20×20 Å) centered around E530, D559, E568 and D604 was calculated using ICMs (Molsoft L.L.C., San Diego, Calif., USA) docking tools.

d. Full flexible ‘ligand’ docking of the 12,782,590 screening molecules (default parameters) was performed using ICM (Molsoft L.L.C., San Diego, Calif., USA).

e. The chemical structures and predicted binding conformations of the top 0.001% scored compounds with MW<500 Da, 0.2<drug-like score<1, clogP<5, number of rotatable bonds<12, compound strain<10 were manually assessed.

f. Initially, a total of 49 high scored compounds were acquired and of those 27 were tested for TONSL ARD binding using a standard native MS binding experiment. A protocol for an MS binding experiment is outlined in Example 10.

Discovery of TONSL-ARD Hits in Primary Binding Screen

Among the compounds tested in competition binding experiments with an unmodified histone H4 peptide tail (SEQ ID NO), weak binding to TONSL ARD (amino acids 512-692 of SEQ ID NO:16) were observed for at least one chemo type based on a “3-[(3-Aminocyclopentyl)carbonyl]-1H-quinolin-4-one” scaffold. AG100021 (MW 317) was the best binding compound in this first binding experiment forming ˜20% complex at 20 μM.

Structural Data

Annex 1—Structure data of TONSL-GFP

The structure data including the coordinates of the “Crystal structure of Human TONSL and MCM2 HBDS binding to a histone H3-H4 tetramer” are provided in the PDB database under the PDB ID 53A4. As used herein the term “PDB ID 5JA4” refers to the PDB ID 5JA4 as deposited with PDB on 11 Apr. 2016. The PDB ID 5JA4 has the DOI: 10.2211/pdb5ja4/pdb, and is accessible at http://www.rcsb.org/pdb/explore/explore.do?structureId=5JA4. The structure data including the same coordinates as PDB ID 5JA4 are also provided in Annex 1 of Danish patent application PA 2015 00605 and in Annex 1 of US patent application 62/324,257.

SEQUENCE DATA SEQ ID NO: 1-TONSL WT/GFP atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgta aacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaag ttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtg cagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggc tacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctg gggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggc atcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgcc ctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatc actctcggcatggacgagctgtacaagggcgcgccaATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGC AAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGGCGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGG GAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCTCTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGG GAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCCCACCGCAAGATCGGAGAGCGCCTGGCCGAGATG GAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAGTACCTGGAGCTGGCACATTCCCTGCGCAACCAC ACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGCACCCACCTGGACATCTATGACCACTGCCAGTCG AGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAGAAGAGCTTGGCTATTGTGGATGAGGAGCTGGAG GGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGGACCCGCCTCTATCTCAACCTGGGCCTCACCTTT GAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTACTTCAGGAAGAGCATCTTCCTTGCGGAGCAGAAC CACCTTTACGAGGACCTATTCCGCGCCCGCTACAACCTGGGCACCATCCACTGGCGCGCGGGCCAGCAC TCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAGTGTGCGCACACCATGAGGAAGCGGTTCATGGAG AGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAAGACCTGGGAGACTTTTTGGCTGCCAAGCGAGCC CTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCTGTGCAGAGGGCAGCCATCTGTCAGAACCTCCAG CATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTGGAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCC ATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCCAAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCT TACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGACAGACCGGGTGCTGAGCGGGCCATCATCCACGTG TCCCTGGCCACCACACTGGGAGACATGAAGGACCACCATGGGGCCGTGCGCCACTATGAGGAGGAACTG AGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAGACCTGGCTGAACATTGCACTGTCCCGCGAGGAG GCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTCCAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAG CGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTCCATACCGTGCAGCTGAGGCTGCAGCCCCAGGAG GCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGTGTAGCTGAAGATGAAGATGAGGAGGAGGAGGCG GAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTGGAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAG GACGACACCGATGGCCTGACCCCGCAGCTGGAGGAGGACGAGGAGCTTCAGGGCCACCTGGGCCGGCGG AAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGGGCGACCCTGCTGCACCGAGCCTGCATCGAGGGC CAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGCCACCCCCTTAACCCTCGGGACTACTGTGGCTGG ACACCTCTGCACGAGGCCTGCAACTACGGGCATCTAGAAATTGTCCGCTTCCTGCTGGACCACGGGGCC CCGCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGCATCACCCCCCTCCACGATGCCCTCAACTGTG GCCACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGGGCGTCCGTCACCCTCCGCACTCGAAAGGGCC TCAGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTGTACCGCAGGGACCTGGACCTGGAGACGCGGC AGAAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCTGCCTCGGGCCAAGATCCCCACAGCTCCCAGG CCTTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAGACCTCTCCTCCTTTGAGCCCCTGCCCAGAAC CCCCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAGGTCCATGTCAGGGTCTCCCCAGGGCAGGCGG CACCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGGCCAGCCAGCAGCAGCAGCAGCTCAGAAGGCG AGGACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGGCCTCGGTGCTCGGCCACAGCACAACGGGTGG CAGCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCAGCCACAGCCAGCACCAGCCGGGCAGCCTACC AGGCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGGCTGGGGCCTGGCCCACCGCGGGGCCACAGCA AAGCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAGGAGTGCCTGGCTGGGGACTGGCTGGAGCTGG ACATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGGGGCACTGGAGACAACCGCAGGCCCAGTAGTA CCTCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCCCGAGCCAAGCAGGTCCGCCTGACCTGCATGC AGAGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGCCTGGCTTCAGAACCTCCAGGGAGCCCCAGCA CCCCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCGGCAGGCCAGCCCTTGGGTCCGGCCCCGCCCC CTCCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTCTTCCTCATCCCTGTCCCACACAGCAGTGACA CCCACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAGCGCTACTACCAGACCTGCGGGCTGCTGCCCA GGCTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCACAGGACCTCATCCCTGATGTGCTGCAGAGCA ATGACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTGCCCCCGTTGACTGACCGCTACCGCAGGGCCT GCCAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTGCAGGCCGTGGAGCTCCAGGGCTTGGGCCTCT CGTTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAGCTTACACCCCTGCTGCGGGCCCTCAAGCTGC ACACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGGCTGGGGGACAAGTGTGTGGCTGAGCTGGTGG CTGCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGACCTCTCCTCCAATCACCTGGGTCCCGAAGGCC TGCGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACCTTGCAGAGTTTGGAGgaattagatctatcga tgaACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCCTCCCTCCTGCACGCCTGCCCCTTACTCAGCA CCCTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTCTTTCTGAGCCACCAGACAGCACTGGGTAGTG CTTTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTGTCCTACAACGCCCTGGGAGCCCCTGCCCTGG CCAGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTGCACTTAGAGCTCAGCTCCGTGGCAGCCGGCA AGGGTGATTCGGACCTCATGGAGCCTGTATTCCGATACCTGGCCAAGGAAGGCTGTGCTCTAGCCCACC TGACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTTAGAGACCTGTGCAGATGTCTCTCTCTGTGCC CCTCACTCATCTCACTGGATCTGTCTGCCAACCCTGAGATCAGCTGTGCCAGCTTGGAAGAGCTCCTGT CCACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTTGGCCTGTCAGGCTGCGCCGTCCAGGGTCCCC TGGGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTCCGGGAACTGCAGCTGTGCAGCAGACGCCTCT GCGCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCCAGTCGGCCGGGCCCCGGCGAGTGCACGCTGG ACCACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 2-TONSL WT ATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGCAAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGG CGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGGGAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCT CTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGGGAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCC CACCGCAAGATCGGAGAGCGCCTGGCCGAGATGGAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAG TACCTGGAGCTGGCACATTCCCTGCGCAACCACACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGC ACCCACCTGGACATCTATGACCACTGCCAGTCGAGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAG AAGAGCTTGGCTATTGTGGATGAGGAGCTGGAGGGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGG ACCCGCCTCTATCTCAACCTGGGCCTCACCTTTGAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTAC TTCAGGAAGAGCATCTTCCTTGCGGAGCAGAACCACCTTTACGAGGACCTATTCCGCGCCCGCTACAAC CTGGGCACCATCCACTGGCGCGCGGGCCAGCACTCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAG TGTGCGCACACCATGAGGAAGCGGTTCATGGAGAGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAA GACCTGGGAGACTTTTTGGCTGCCAAGCGAGCCCTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCT GTGCAGAGGGCAGCCATCTGTCAGAACCTCCAGCATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTG GAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCCATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCC AAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCTTACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGAC AGACCGGGTGCTGAGCGGGCCATCATCCACGTGTCCCTGGCCACCACACTGGGAGACATGAAGGACCAC CATGGGGCCGTGCGCCACTATGAGGAGGAACTGAGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAG ACCTGGCTGAACATTGCACTGTCCCGCGAGGAGGCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTC CAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAGCGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTC CATACCGTGCAGCTGAGGCTGCAGCCCCAGGAGGCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGT GTAGCTGAAGATGAAGATGAGGAGGAGGAGGCGGAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTG GAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAGGACGACACCGATGGCCTGACCCCGCAGCTGGAGGAG GACGAGGAGCTTCAGGGCCACCTGGGCCGGCGGAAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGG GCGACCCTGCTGCACCGAGCCTGCATCGAGGGCCAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGC CACCCCCTTAACCCTCGGGACTACTGTGGCTGGACACCTCTGCACGAGGCCTGCAACTACGGGCATCTA GAAATTGTCCGCTTCCTGCTGGACCACGGGGCCGCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGC ATCACCCCCCTCCACGATGCCCTCAACTGTGGCCACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGG GCGTCCGTCACCCTCCGCACTCGAAAGGGCCTCAGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTG TACCGCAGGGACCTGGACCTGGAGACGCGGCAGAAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCT GCCTCGGGCCAAGATCCCCACAGCTCCCAGGCCTTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAG ACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCCCCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAG GTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCACCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGG CCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAGGACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGG CCTCGGTGCTCGGCCACAGCACAACGGGTGGCAGCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCA GCCACAGCCAGCACCAGCCGGGCAGCCTACCAGGCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGG CTGGGGCCTGGCCCACCGCGGGGCCACAGCAAAGCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAG GAGTGCCTGGCTGGGGACTGGCTGGAGCTGGACATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGG GGCACTGGAGACAACCGCAGGCCCAGTAGTACCTCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCC CGAGCCAAGCAGGTCCGCCTGACCTGCATGCAGAGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGC CTGGCTTCAGAACCTCCAGGGAGCCCCAGCACCCCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCG GCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCTCCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTC TTCCTCATCCCTGTCCCACACAGCAGTGACACCCACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAG CGCTACTACCAGACCTGCGGGCTGCTGCCCAGGCTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCA CAGGACCTCATCCCTGATGTGCTGCAGAGCAATGACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTG CCCCCGTTGACTGACCGCTACCGCAGGGCCTGCCAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTG CAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCGTTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAG CTTACACCCCTGCTGCGGGCCCTCAAGCTGCACACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGG CTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCTGCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGAC CTCTCCTCCAATCACCTGGGTCCCGAAGGCCTGCGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACC TTGCAGAGTTTGGAGgaattagatctatcgatgaACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCC TCCCTCCTGCACGCCTGCCCCTTACTCAGCACCCTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTC TTTCTGAGCCACCAGACAGCACTGGGTAGTGCTTTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTG TCCTACAACGCCCTGGGAGCCCCTGCCCTGGCCAGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTG CACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAGGGTGATTCGGACCTCATGGAGCCTGTATTCCGATAC CTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTGACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTT AGAGACCTGTGCAGATGTCTCTCTCTGTGCCCCTCACTCATCTCACTGGATCTGTCTGCCAACCCTGAG ATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCCACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTT GGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTGGGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTC CGGGAACTGCAGCTGTGCAGCAGACGCCTCTGCGCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCC AGTCGGCCGGGCCCCGGCGAGTGCACGCTGGACCACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 3-TONSL E530A ATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGCAAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGG CGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGGGAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCT CTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGGGAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCC CACCGCAAGATCGGAGAGCGCCTGGCCGAGATGGAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAG TACCTGGAGCTGGCACATTCCCTGCGCAACCACACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGC ACCCACCTGGACATCTATGACCACTGCCAGTCGAGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAG AAGAGCTTGGCTATTGTGGATGAGGAGCTGGAGGGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGG ACCCGCCTCTATCTCAACCTGGGCCTCACCTTTGAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTAC TTCAGGAAGAGCATCTTCCTTGCGGAGCAGAACCACCTTTACGAGGACCTATTCCGCGCCCGCTACAAC CTGGGCACCATCCACTGGCGCGCGGGCCAGCACTCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAG TGTGCGCACACCATGAGGAAGCGGTTCATGGAGAGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAA GACCTGGGAGACTTTTTGGCTGCCAAGCGAGCCCTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCT GTGCAGAGGGCAGCCATCTGTCAGAACCTCCAGCATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTG GAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCCATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCC AAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCTTACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGAC AGACCGGGTGCTGAGCGGGCCATCATCCACGTGTCCCTGGCCACCACACTGGGAGACATGAAGGACCAC CATGGGGCCGTGCGCCACTATGAGGAGGAACTGAGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAG ACCTGGCTGAACATTGCACTGTCCCGCGAGGAGGCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTC CAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAGCGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTC CATACCGTGCAGCTGAGGCTGCAGCCCCAGGAGGCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGT GTAGCTGAAGATGAAGATGAGGAGGAGGAGGCGGAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTG GAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAGGACGACACCGATGGCCTGACCCCGCAGCTGGAGGAG GACGAGGAGCTTCAGGGCCACCTGGGCCGGCGGAAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGG GCGACCCTGCTGCACCGAGCCTGCATCGAGGGCCAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGC CACCCCCTTAACCCTCGGGACTACTGTGGCTGGACACCTCTGCACGAGGCCTGCAACTACGGGCATCTA GAAATTGTCCGCTTCCTGCTGGACCACGGGGCCGCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGC ATCACCCCCCTCCACGATGCCCTCAACTGTGGCCACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGG GCGTCCGTCACCCTCCGCACTCGAAAGGGCCTCAGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTG TACCGCAGGGACCTGGACCTGGAGACGCGGCAGAAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCT GCCTCGGGCCAAGATCCCCACAGCTCCCAGGCCTTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAG ACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCCCCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAG GTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCACCAGCCATGGCCAGGCTGCGGAGGAGCAGGCATGGG CCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAGGACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGG CCTCGGTGCTCGGCCACAGCACAACGGGTGGCAGCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCA GCCACAGCCAGCACCAGCCGGGCAGCCTACCAGGCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGG CTGGGGCCTGGCCCACCGCGGGGCCACAGCAAAGCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAG GAGTGCCTGGCTGGGGACTGGCTGGAGCTGGACATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGG GGCACTGGAGACAACCGCAGGCCCAGTAGTACCTCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCC CGAGCCAAGCAGGTCCGCCTGACCTGCATGCAGAGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGC CTGGCTTCAGAACCTCCAGGGAGCCCCAGCACCCCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCG GCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCTCCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTC TTCCTCATCCCTGTCCCACACAGCAGTGACACCCACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAG CGCTACTACCAGACCTGCGGGCTGCTGCCCAGGCTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCA CAGGACCTCATCCCTGATGTGCTGCAGAGCAATGACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTG CCCCCGTTGACTGACCGCTACCGCAGGGCCTGCCAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTG CAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCGTTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAG CTTACACCCCTGCTGCGGGCCCTCAAGCTGCACACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGG CTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCTGCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGAC CTCTCCTCCAATCACCTGGGTCCCGAAGGCCTGCGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACC TTGCAGAGTTTGGAGgaattagatctatcgatgaACCCCCTGGGGGACGCCTGTGGCCAGTCCCTGGCC TCCCTCCTGCACGCCTGCCCCTTACTCAGCACCCTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTC TTTCTGAGCCACCAGACAGCACTGGGTAGTGCTTTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTG TCCTACAACGCCCTGGGAGCCCCTGCCCTGGCCAGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTG CACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAGGGTGATTCGGACCTCATGGAGCCTGTATTCCGATAC CTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTGACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTT AGAGACCTGTGCAGATGTCTCTCTCTGTGCCCCTCACTCATCTCACTGGATCTGTCTGCCAACCCTGAG ATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCCACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTT GGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTGGGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTC CGGGAACTGCAGCTGTGCAGCAGACGCCTCTGCGCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCC AGTCGGCCGGGCCCCGGCGAGTGCACGCTGGACCACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 4-TONSL E530A/GFP atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgta aacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaag ttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtg cagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggc tacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctg gggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggc atcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgcc ctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatc actctcggcatggacgagctgtacaagggcgcgccaATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGC AAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGGCGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGG GAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCTCTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGG GAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCCCACCGCAAGATCGGAGAGCGCCTGGCCGAGATG GAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAGTACCTGGAGCTGGCACATTCCCTGCGCAACCAC ACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGCACCCACCTGGACATCTATGACCACTGCCAGTCG AGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAGAAGAGCTTGGCTATTGTGGATGAGGAGCTGGAG GGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGGACCCGCCTCTATCTCAACCTGGGCCTCACCTTT GAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTACTTCAGGAAGAGCATCTTCCTTGCGGAGCAGAAC CACCTTTACGAGGACCTATTCCGCGCCCGCTACAACCTGGGCACCATCCACTGGCGCGCGGGCCAGCAC TCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAGTGTGCGCACACCATGAGGAAGCGGTTCATGGAG AGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAAGACCTGGGAGACTTTTTGGCTGCCAAGCGAGCC CTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCTGTGCAGAGGGCAGCCATCTGTCAGAACCTCCAG CATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTGGAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCC ATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCCAAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCT TACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGACAGACCGGGTGCTGAGCGGGCCATCATCCACGTG TCCCTGGCCACCACACTGGGAGACATGAAGGACCACCATGGGGCCGTGCGCCACTATGAGGAGGAACTG AGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAGACCTGGCTGAACATTGCACTGTCCCGCGAGGAG GCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTCCAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAG CGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTCCATACCGTGCAGCTGAGGCTGCAGCCCCAGGAG GCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGTGTAGCTGAAGATGAAGATGAGGAGGAGGAGGCG GAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTGGAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAG GACGACACCGATGGCCTGACCCCGCAGCTGGAGGAGGACGAGGAGCTTCAGGGCCACCTGGGCCGGCGG AAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGGGCGACCCTGCTGCACCGAGCCTGCATCGAGGGC CAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGCCACCCCCTTAACCCTCGGGACTACTGTGGCTGG ACACCTCTGCACGAGGCCTGCAACTACGGGCATCTAGAAATTGTCCGCTTCCTGCTGGACCACGGGGCC GCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGCATCACCCCCCTCCACGATGCCCTCAACTGTGGC CACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGGGCGTCCGTCACCCTCCGCACTCGAAAGGGCCTC AGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTGTACCGCAGGGACCTGGACCTGGAGACGCGGCAG AAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCTGCCTCGGGCCAAGATCCCCACAGCTCCCAGGCC TTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAGACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCC CCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAGGTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCA CCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGGCCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAG GACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGGCCTCGGTGCTCGGCCACAGCACAACGGGTGGCA GCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCAGCCACAGCCAGCACCAGCCGGGCAGCCTACCAG GCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGGCTGGGGCCTGGCCCACCGCGGGGCCACAGCAAA GCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAGGAGTGCCTGGCTGGGGACTGGCTGGAGCTGGAC ATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGGGGCACTGGAGACAACCGCAGGCCCAGTAGTACC TCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCCCGAGCCAAGCAGGTCCGCCTGACCTGCATGCAG AGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGCCTGGCTTCAGAACCTCCAGGGAGCCCCAGCACC CCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCGGCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCT CCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTCTTCCTCATCCCTGTCCCACACAGCAGTGACACC CACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAGCGCTACTACCAGACCTGCGGGCTGCTGCCCAGG CTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCACAGGACCTCATCCCTGATGTGCTGCAGAGCAAT GACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTGCCCCCGTTGACTGACCGCTACCGCAGGGCCTGC CAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTGCAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCG TTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAGCTTACACCCCTGCTGCGGGCCCTCAAGCTGCAC ACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGGCTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCT GCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGACCTCTCCTCCAATCACCTGGGTCCCGAAGGCCTG CGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACCTTGCAGAGTTTGGAGgaattagatctatcgatg aACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCCTCCCTCCTGCACGCCTGCCCCTTACTCAGCACC CTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTCTTTCTGAGCCACCAGACAGCACTGGGTAGTGCT TTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTGTCCTACAACGCCCTGGGAGCCCCTGCCCTGGCC AGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTGCACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAG GGTGATTCGGACCTCATGGAGCCTGTATTCCGATACCTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTG ACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTTAGAGACCTGTGCAGATGTCTCTCTCTGTGCCCC TCACTCATCTCACTGGATCTGTCTGCCAACCCTGAGATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCC ACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTTGGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTG GGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTCCGGGAACTGCAGCTGTGCAGCAGACGCCTCTGC GCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCCAGTCGGCCGGGCCCCGGCGAGTGCACGCTGGAC CACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 5-TONSL D559A ATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGCAAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGG CGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGGGAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCT CTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGGGAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCC CACCGCAAGATCGGAGAGCGCCTGGCCGAGATGGAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAG TACCTGGAGCTGGCACATTCCCTGCGCAACCACACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGC ACCCACCTGGACATCTATGACCACTGCCAGTCGAGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAG AAGAGCTTGGCTATTGTGGATGAGGAGCTGGAGGGGACACTGGCCCAGGGAAAGCTGAATGAGATGAGG ACCCGCCTCTATCTCAACCTGGGCCTCACCTTTGAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTAC TTCAGGAAGAGCATCTTCCTTGCGGAGCAGAACCACCTTTACGAGGACCTATTCCGCGCCCGCTACAAC CTGGGCACCATCCACTGGCGCGCGGGCCAGCACTCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAG TGTGCGCACACCATGAGGAAGCGGTTCATGGAGAGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAA GACCTGGGAGACTTTTTGGCTGCCAAGCGAGCCCTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCT GTGCAGAGGGCAGCCATCTGTCAGAACCTCCAGCATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTG GAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCCATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCC AAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCTTACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGAC AGACCGGGTGCTGAGCGGGCCATCATCCACGTGTCCCTGGCCACCACACTGGGAGACATGAAGGACCAC CATGGGGCCGTGCGCCACTATGAGGAGGAACTGAGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAG ACCTGGCTGAACATTGCACTGTCCCGCGAGGAGGCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTC CAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAGCGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTC CATACCGTGCAGCTGAGGCTGCAGCCCCAGGAGGCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGT GTAGCTGAAGATGAAGATGAGGAGGAGGAGGCGGAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTG GAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAGGACGACACCGATGGCCTGACCCCGCAGCTGGAGGAG GACGAGGAGCTTCAGGGCCACCTGGGCCGGCGGAAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGG GAGACCCTGCTGCACCGAGCCTGCATCGAGGGCCAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGC CACCCCCTTAACCCTCGGGCCTACTGTGGCTGGACACCTCTGCACGAGGCCTGCAACTACGGGCATCTA GAAATTGTCCGCTTCCTGCTGGACCACGGGGCCGCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGC ATCACCCCCCTCCACGATGCCCTCAACTGTGGCCACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGG GCGTCCGTCACCCTCCGCACTCGAAAGGGCCTCAGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTG TACCGCAGGGACCTGGACCTGGAGACGCGGCAGAAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCT GCCTCGGGCCAAGATCCCCACAGCTCCCAGGCCTTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAG ACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCCCCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAG GTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCACCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGG CCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAGGACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGG CCTCGGTGCTCGGCCACAGCACAACGGGTGGCAGCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCA GCCACAGCCAGCACCAGCCGGGCAGCCTACCAGGCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGG CTGGGGCCTGGCCCACCGCGGGGCCACAGCAAAGCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAG GAGTGCCTGGCTGGGGACTGGCTGGAGCTGGACATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGG GGCACTGGAGACAACCGCAGGCCCAGTAGTACCTCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCC CGAGCCAAGCAGGTCCGCCTGACCTGCATGCAGAGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGC CTGGCTTCAGAACCTCCAGGGAGCCCCAGCACCCCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCG GCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCTCCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTC TTCCTCATCCCTGTCCCACACAGCAGTGACACCCACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAG CGCTACTACCAGACCTGCGGGCTGCTGCCCAGGCTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCA CAGGACCTCATCCCTGATGTGCTGCAGAGCAATGACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTG CCCCCGTTGACTGACCGCTACCGCAGGGCCTGCCAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTG CAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCGTTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAG CTTACACCCCTGCTGCGGGCCCTCAAGCTGCACACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGG CTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCTGCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGAC CTCTCCTCCAATCACCTGGGTCCCGAAGGCCTGCGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACC TTGCAGAGTTTGGAGgaattagatctatcgatgaACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCC TCCCTCCTGCACGCCTGCCCCTTACTCAGCACCCTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTC TTTCTGAGCCACCAGACAGCACTGGGTAGTGCTTTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTG TCCTACAACGCCCTGGGAGCCCCTGCCCTGGCCAGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTG CACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAGGGTGATTCGGACCTCATGGAGCCTGTATTCCGATAC CTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTGACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTT AGAGACCTGTGCAGATGTCTCTCTCTGTGCCCCTCACTCATCTCACTGGATCTGTCTGCCAACCCTGAG ATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCCACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTT GGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTGGGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTC CGGGAACTGCAGCTGTGCAGCAGACGCCTCTGCGCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCC AGTCGGCCGGGCCCCGGCGAGTGCACGCTGGACCACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 6-TONSL D559A/GFP atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgta aacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaag ttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtg cagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggc tacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctg gggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggc atcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgcc ctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatc actctcggcatggacgagctgtacaagggcgcgccaATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGC AAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGGCGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGG GAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCTCTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGG GAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCCCACCGCAAGATCGGAGAGCGCCTGGCCGAGATG GAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAGTACCTGGAGCTGGCACATTCCCTGCGCAACCAC ACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGCACCCACCTGGACATCTATGACCACTGCCAGTCG AGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAGAAGAGCTTGGCTATTGTGGATGAGGAGCTGGAG GGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGGACCCGCCTCTATCTCAACCTGGGCCTCACCTTT GAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTACTTCAGGAAGAGCATCTTCCTTGCGGAGCAGAAC CACCTTTACGAGGACCTATTCCGCGCCCGCTACAACCTGGGCACCATCCACTGGCGCGCGGGCCAGCAC TCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAGTGTGCGCACACCATGAGGAAGCGGTTCATGGAG AGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAAGACCTGGGAGACTTTTTGGCTGCCAAGCGAGCC CTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCTGTGCAGAGGGCAGCCATCTGTCAGAACCTCCAG CATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTGGAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCC ATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCCAAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCT TACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGACAGACCGGGTGCTGAGCGGGCCATCATCCACGTG TCCCTGGCCACCACACTGGGAGACATGAAGGACCACCATGGGGCCGTGCGCCACTATGAGGAGGAACTG AGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAGACCTGGCTGAACATTGCACTGTCCCGCGAGGAG GCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTCCAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAG CGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTCCATACCGTGCAGCTGAGGCTGCAGCCCCAGGAG GCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGTGTAGCTGAAGATGAAGATGAGGAGGAGGAGGCG GAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTGGAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAG GACGACACCGATGGCCTGACCCCGCAGCTGGAGGAGGACGAGGAGCTTCAGGGCCACCTGGGCCGGCGG AAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGGGAGACCCTGCTGCACCGAGCCTGCATCGAGGGC CAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGCCACCCCCTTAACCCTCGGGCCTACTGTGGCTGG ACACCTCTGCACGAGGCCTGCAACTACGGGCATCTAGAAATTGTCCGCTTCCTGCTGGACCACGGGGCC GCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGCATCACCCCCCTCCACGATGCCCTCAACTGTGGC CACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGGGCGTCCGTCACCCTCCGCACTCGAAAGGGCCTC AGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTGTACCGCAGGGACCTGGACCTGGAGACGCGGCAG AAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCTGCCTCGGGCCAAGATCCCCACAGCTCCCAGGCC TTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAGACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCC CCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAGGTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCA CCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGGCCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAG GACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGGCCTCGGTGCTCGGCCACAGCACAACGGGTGGCA GCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCAGCCACAGCCAGCACCAGCCGGGCAGCCTACCAG GCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGGCTGGGGCCTGGCCCACCGCGGGGCCACAGCAAA GCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAGGAGTGCCTGGCTGGGGACTGGCTGGAGCTGGAC ATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGGGGCACTGGAGACAACCGCAGGCCCAGTAGTACC TCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCCCGAGCCAAGCAGGTCCGCCTGACCTGCATGCAG AGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGCCTGGCTTCAGAACCTCCAGGGAGCCCCAGCACC CCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCGGCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCT CCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTCTTCCTCATCCCTGTCCCACACAGCAGTGACACC CACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAGCGCTACTACCAGACCTGCGGGCTGCTGCCCAGG CTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCACAGGACCTCATCCCTGATGTGCTGCAGAGCAAT GACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTGCCCCCGTTGACTGACCGCTACCGCAGGGCCTGC CAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTGCAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCG TTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAGCTTACACCCCTGCTGCGGGCCCTCAAGCTGCAC ACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGGCTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCT GCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGACCTCTCCTCCAATCACCTGGGTCCCGAAGGCCTG CGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACCTTGCAGAGTTTGGAGgaattagatctatcgatg aACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCCTCCCTCCTGCACGCCTGCCCCTTACTCAGCACC CTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTCTTTCTGAGCCACCAGACAGCACTGGGTAGTGCT TTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTGTCCTACAACGCCCTGGGAGCCCCTGCCCTGGCC AGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTGCACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAG GGTGATTCGGACCTCATGGAGCCTGTATTCCGATACCTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTG ACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTTAGAGACCTGTGCAGATGTCTCTCTCTGTGCCCC TCACTCATCTCACTGGATCTGTCTGCCAACCCTGAGATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCC ACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTTGGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTG GGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTCCGGGAACTGCAGCTGTGCAGCAGACGCCTCTGC GCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCCAGTCGGCCGGGCCCCGGCGAGTGCACGCTGGAC CACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 7-TONSL W563A ATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGCAAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGG CGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGGGAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCT CTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGGGAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCC CACCGCAAGATCGGAGAGCGCCTGGCCGAGATGGAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAG TACCTGGAGCTGGCACATTCCCTGCGCAACCACACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGC ACCCACCTGGACATCTATGACCACTGCCAGTCGAGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAG AAGAGCTTGGCTATTGTGGATGAGGAGCTGGAGGGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGG ACCCGCCTCTATCTCAACCTGGGCCTCACCTTTGAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTAC TTCAGGAAGAGCATCTTCCTTGCGGAGCAGAACCACCTTTACGAGGACCTATTCCGCGCCCGCTACAAC CTGGGCACCATCCACTGGCGCGCGGGCCAGCACTCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAG TGTGCGCACACCATGAGGAAGCGGTTCATGGAGAGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAA GACCTGGGAGACTTTTTGGCTGCCAAGCGAGCCCTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCT GTGCAGAGGGCAGCCATCTGTCAGAACCTCCAGCATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTG GAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCCATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCC AAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCTTACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGAC AGACCGGGTGCTGAGCGGGCCATCATCCACGTGTCCCTGGCCACCACACTGGGAGACATGAAGGACCAC CATGGGGCCGTGCGCCACTATGAGGAGGAACTGAGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAG ACCTGGCTGAACATTGCACTGTCCCGCGAGGAGGCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTC CAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAGCGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTC CATACCGTGCAGCTGAGGCTGCAGCCCCAGGAGGCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGT GTAGCTGAAGATGAAGATGAGGAGGAGGAGGCGGAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTG GAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAGGACGACACCGATGGCCTGACCCCGCAGCTGGAGGAG GACGAGGAGCTTCAGGGCCACCTGGGCCGGCGGAAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGG GAGACCCTGCTGCACCGAGCCTGCATCGAGGGCCAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGC CACCCCCTTAACCCTCGGGACTACTGTGGCGCGACACCTCTGCACGAGGCCTGCAACTACGGGCATCTA GAAATTGTCCGCTTCCTGCTGGACCACGGGGCCGCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGC ATCACCCCCCTCCACGATGCCCTCAACTGTGGCCACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGG GCGTCCGTCACCCTCCGCACTCGAAAGGGCCTCAGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTG TACCGCAGGGACCTGGACCTGGAGACGCGGCAGAAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCT GCCTCGGGCCAAGATCCCCACAGCTCCCAGGCCTTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAG ACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCCCCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAG GTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCACCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGG CCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAGGACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGG CCTCGGTGCTCGGCCACAGCACAACGGGTGGCAGCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCA GCCACAGCCAGCACCAGCCGGGCAGCCTACCAGGCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGG CTGGGGCCTGGCCCACCGCGGGGCCACAGCAAAGCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAG GAGTGCCTGGCTGGGGACTGGCTGGAGCTGGACATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGG GGCACTGGAGACAACCGCAGGCCCAGTAGTACCTCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCC CGAGCCAAGCAGGTCCGCCTGACCTGCATGCAGAGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGC CTGGCTTCAGAACCTCCAGGGAGCCCCAGCACCCCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCG GCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCTCCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTC TTCCTCATCCCTGTCCCACACAGCAGTGACACCCACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAG CGCTACTACCAGACCTGCGGGCTGCTGCCCAGGCTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCA CAGGACCTCATCCCTGATGTGCTGCAGAGCAATGACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTG CCCCCGTTGACTGACCGCTACCGCAGGGCCTGCCAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTG CAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCGTTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAG CTTACACCCCTGCTGCGGGCCCTCAAGCTGCACACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGG CTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCTGCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGAC CTCTCCTCCAATCACCTGGGTCCCGAAGGCCTGCGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACC TTGCAGAGTTTGGAGgaattagatctatcgatgaACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCC TCCCTCCTGCACGCCTGCCCCTTACTCAGCACCCTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTC TTTCTGAGCCACCAGACAGCACTGGGTAGTGCTTTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTG TCCTACAACGCCCTGGGAGCCCCTGCCCTGGCCAGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTG CACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAGGGTGATTCGGACCTCATGGAGCCTGTATTCCGATAC CTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTGACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTT AGAGACCTGTGCAGATGTCTCTCTCTGTGCCCCTCACTCATCTCACTGGATCTGTCTGCCAACCCTGAG ATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCCACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTT GGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTGGGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTC CGGGAACTGCAGCTGTGCAGCAGACGCCTCTGCGCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCC AGTCGGCCGGGCCCCGGCGAGTGCACGCTGGACCACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 8-TONSL W563A/GFP atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgta aacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaag ttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtg cagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggc tacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctg gggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggc atcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgcc ctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatc actctcggcatggacgagctgtacaagggcgcgccaATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGC AAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGGCGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGG GAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCTCTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGG GAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCCCACCGCAAGATCGGAGAGCGCCTGGCCGAGATG GAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAGTACCTGGAGCTGGCACATTCCCTGCGCAACCAC ACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGCACCCACCTGGACATCTATGACCACTGCCAGTCG AGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAGAAGAGCTTGGCTATTGTGGATGAGGAGCTGGAG GGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGGACCCGCCTCTATCTCAACCTGGGCCTCACCTTT GAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTACTTCAGGAAGAGCATCTTCCTTGCGGAGCAGAAC CACCTTTACGAGGACCTATTCCGCGCCCGCTACAACCTGGGCACCATCCACTGGCGCGCGGGCCAGCAC TCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAGTGTGCGCACACCATGAGGAAGCGGTTCATGGAG AGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAAGACCTGGGAGACTTTTTGGCTGCCAAGCGAGCC CTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCTGTGCAGAGGGCAGCCATCTGTCAGAACCTCCAG CATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTGGAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCC ATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCCAAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCT TACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGACAGACCGGGTGCTGAGCGGGCCATCATCCACGTG TCCCTGGCCACCACACTGGGAGACATGAAGGACCACCATGGGGCCGTGCGCCACTATGAGGAGGAACTG AGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAGACCTGGCTGAACATTGCACTGTCCCGCGAGGAG GCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTCCAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAG CGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTCCATACCGTGCAGCTGAGGCTGCAGCCCCAGGAG GCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGTGTAGCTGAAGATGAAGATGAGGAGGAGGAGGCG GAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTGGAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAG GACGACACCGATGGCCTGACCCCGCAGCTGGAGGAGGACGAGGAGCTTCAGGGCCACCTGGGCCGGCGG AAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGGGAGACCCTGCTGCACCGAGCCTGCATCGAGGGC CAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGCCACCCCCTTAACCCTCGGGACTACTGTGGCGCG ACACCTCTGCACGAGGCCTGCAACTACGGGCATCTAGAAATTGTCCGCTTCCTGCTGGACCACGGGGCC GCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGCATCACCCCCCTCCACGATGCCCTCAACTGTGGC CACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGGGCGTCCGTCACCCTCCGCACTCGAAAGGGCCTC AGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTGTACCGCAGGGACCTGGACCTGGAGACGCGGCAG AAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCTGCCTCGGGCCAAGATCCCCACAGCTCCCAGGCC TTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAGACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCC CCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAGGTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCA CCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGGCCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAG GACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGGCCTCGGTGCTCGGCCACAGCACAACGGGTGGCA GCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCAGCCACAGCCAGCACCAGCCGGGCAGCCTACCAG GCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGGCTGGGGCCTGGCCCACCGCGGGGCCACAGCAAA GCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAGGAGTGCCTGGCTGGGGACTGGCTGGAGCTGGAC ATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGGGGCACTGGAGACAACCGCAGGCCCAGTAGTACC TCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCCCGAGCCAAGCAGGTCCGCCTGACCTGCATGCAG AGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGCCTGGCTTCAGAACCTCCAGGGAGCCCCAGCACC CCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCGGCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCT CCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTCTTCCTCATCCCTGTCCCACACAGCAGTGACACC CACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAGCGCTACTACCAGACCTGCGGGCTGCTGCCCAGG CTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCACAGGACCTCATCCCTGATGTGCTGCAGAGCAAT GACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTGCCCCCGTTGACTGACCGCTACCGCAGGGCCTGC CAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTGCAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCG TTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAGCTTACACCCCTGCTGCGGGCCCTCAAGCTGCAC ACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGGCTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCT GCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGACCTCTCCTCCAATCACCTGGGTCCCGAAGGCCTG CGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACCTTGCAGAGTTTGGAGgaattagatctatcgatg aACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCCTCCCTCCTGCACGCCTGCCCCTTACTCAGCACC CTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTCTTTCTGAGCCACCAGACAGCACTGGGTAGTGCT TTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTGTCCTACAACGCCCTGGGAGCCCCTGCCCTGGCC AGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTGCACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAG GGTGATTCGGACCTCATGGAGCCTGTATTCCGATACCTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTG ACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTTAGAGACCTGTGCAGATGTCTCTCTCTGTGCCCC TCACTCATCTCACTGGATCTGTCTGCCAACCCTGAGATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCC ACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTTGGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTG GGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTCCGGGAACTGCAGCTGTGCAGCAGACGCCTCTGC GCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCCAGTCGGCCGGGCCCCGGCGAGTGCACGCTGGAC CACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 9-TONSL E568A ATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGCAAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGG CGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGGGAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCT CTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGGGAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCC CACCGCAAGATCGGAGAGCGCCTGGCCGAGATGGAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAG TACCTGGAGCTGGCACATTCCCTGCGCAACCACACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGC ACCCACCTGGACATCTATGACCACTGCCAGTCGAGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAG AAGAGCTTGGCTATTGTGGATGAGGAGCTGGAGGGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGG ACCCGCCTCTATCTCAACCTGGGCCTCACCTTTGAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTAC TTCAGGAAGAGCATCTTCCTTGCGGAGCAGAACCACCTTTACGAGGACCTATTCCGCGCCCGCTACAAC CTGGGCACCATCCACTGGCGCGCGGGCCAGCACTCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAG TGTGCGCACACCATGAGGAAGCGGTTCATGGAGAGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAA GACCTGGGAGACTTTTTGGCTGCCAAGCGAGCCCTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCT GTGCAGAGGGCAGCCATCTGTCAGAACCTCCAGCATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTG GAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCCATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCC AAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCTTACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGAC AGACCGGGTGCTGAGCGGGCCATCATCCACGTGTCCCTGGCCACCACACTGGGAGACATGAAGGACCAC CATGGGGCCGTGCGCCACTATGAGGAGGAACTGAGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAG ACCTGGCTGAACATTGCACTGTCCCGCGAGGAGGCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTC CAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAGCGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTC CATACCGTGCAGCTGAGGCTGCAGCCCCAGGAGGCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGT GTAGCTGAAGATGAAGATGAGGAGGAGGAGGCGGAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTG GAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAGGACGACACCGATGGCCTGACCCCGCAGCTGGAGGAG GACGAGGAGCTTCAGGGCCACCTGGGCCGGCGGAAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGG GAGACCCTGCTGCACCGAGCCTGCATCGAGGGCCAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGC CACCCCCTTAACCCTCGGGACTACTGTGGCTGGACACCTCTGCACGCGGCCTGCAACTACGGGCATCTA GAAATTGTCCGCTTCCTGCTGGACCACGGGGCCGCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGC ATCACCCCCCTCCACGATGCCCTCAACTGTGGCCACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGG GCGTCCGTCACCCTCCGCACTCGAAAGGGCCTCAGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTG TACCGCAGGGACCTGGACCTGGAGACGCGGCAGAAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCT GCCTCGGGCCAAGATCCCCACAGCTCCCAGGCCTTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAG ACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCCCCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAG GTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCACCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGG CCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAGGACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGG CCTCGGTGCTCGGCCACAGCACAACGGGTGGCAGCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCA GCCACAGCCAGCACCAGCCGGGCAGCCTACCAGGCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGG CTGGGGCCTGGCCCACCGCGGGGCCACAGCAAAGCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAG GAGTGCCTGGCTGGGGACTGGCTGGAGCTGGACATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGG GGCACTGGAGACAACCGCAGGCCCAGTAGTACCTCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCC CGAGCCAAGCAGGTCCGCCTGACCTGCATGCAGAGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGC CTGGCTTCAGAACCTCCAGGGAGCCCCAGCACCCCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCG GCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCTCCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTC TTCCTCATCCCTGTCCCACACAGCAGTGACACCCACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAG CGCTACTACCAGACCTGCGGGCTGCTGCCCAGGCTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCA CAGGACCTCATCCCTGATGTGCTGCAGAGCAATGACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTG CCCCCGTTGACTGACCGCTACCGCAGGGCCTGCCAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTG CAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCGTTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAG CTTACACCCCTGCTGCGGGCCCTCAAGCTGCACACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGG CTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCTGCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGAC CTCTCCTCCAATCACCTGGGTCCCGAAGGCCTGCGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACC TTGCAGAGTTTGGAGgaattagatctatcgatgaACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCC TCCCTCCTGCACGCCTGCCCCTTACTCAGCACCCTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTC TTTCTGAGCCACCAGACAGCACTGGGTAGTGCTTTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTG TCCTACAACGCCCTGGGAGCCCCTGCCCTGGCCAGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTG CACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAGGGTGATTCGGACCTCATGGAGCCTGTATTCCGATAC CTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTGACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTT AGAGACCTGTGCAGATGTCTCTCTCTGTGCCCCTCACTCATCTCACTGGATCTGTCTGCCAACCCTGAG ATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCCACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTT GGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTGGGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTC CGGGAACTGCAGCTGTGCAGCAGACGCCTCTGCGCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCC AGTCGGCCGGGCCCCGGCGAGTGCACGCTGGACCACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 10-TONSL E568A/GFP atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgta aacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaag ttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtg cagtgcctcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggc tacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctg gggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggc atcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgcc ctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatc actctcggcatggacgagctgtacaagggcgcgccaATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGC AAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGGCGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGG GAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCCATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCC AAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCTTACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGAC AGACCGGGTGCTGAGCGGGCCATCATCCACGTGTCCCTGGCCACCACACTGGGAGACATGAAGGACCAC CATGGGGCCGTGCGCCACTATGAGGAGGAACTGAGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAG ACCTGGCTGAACATTGCACTGTCCCGCGAGGAGGCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTC CAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAGCGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTC CATACCGTGCAGCTGAGGCTGCAGCCCCAGGAGGCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGT GTAGCTGAAGATGAAGATGAGGAGGAGGAGGCGGAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTG GAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAGGACGACACCGATGGCCTGACCCCGCAGCTGGAGGAG GACGAGGAGCTTCAGGGCCACCTGGGCCGGCGGAAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGG GAGACCCTGCTGCACCGAGCCTGCATCGAGGGCCAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGC CACCCCCTTAACCCTCGGGACTACTGTGGCTGGACACCTCTGCACGCGGCCTGCAACTACGGGCATCTA GAAATTGTCCGCTTCCTGCTGGACCACGGGGCCGCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGC ATCACCCCCCTCCACGATGCCCTCAACTGTGGCCACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGG GCGTCCGTCACCCTCCGCACTCGAAAGGGCCTCAGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTG TACCGCAGGGACCTGGACCTGGAGACGCGGCAGAAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCT GCCTCGGGCCAAGATCCCCACAGCTCCCAGGCCTTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAG ACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCCCCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAG GTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCACCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGG CCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAGGACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGG CCTCGGTGCTCGGCCACAGCACAACGGGTGGCAGCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCA GCCACAGCCAGCACCAGCCGGGCAGCCTACCAGGCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGG CTGGGGCCTGGCCCACCGCGGGGCCACAGCAAAGCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAG GAGTGCCTGGCTGGGGACTGGCTGGAGCTGGACATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGG GGCACTGGAGACAACCGCAGGCCCAGTAGTACCTCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCC CGAGCCAAGCAGGTCCGCCTGACCTGCATGCAGAGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGC CTGGCTTCAGAACCTCCAGGGAGCCCCAGCACCCCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCG GCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCTCCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTC TTCCTCATCCCTGTCCCACACAGCAGTGACACCCACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAG CGCTACTACCAGACCTGCGGGCTGCTGCCCAGGCTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCA CAGGACCTCATCCCTGATGTGCTGCAGAGCAATGACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTG CCCCCGTTGACTGACCGCTACCGCAGGGCCTGCCAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTG CAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCGTTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAG CTTACACCCCTGCTGCGGGCCCTCAAGCTGCACACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGG CTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCTGCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGAC CTCTCCTCCAATCACCTGGGTCCCGAAGGCCTGCGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACC TTGCAGAGTTTGGAGgaattagatctatcgatgaACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCC TCCCTCCTGCACGCCTGCCCCTTACTCAGCACCCTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTC TTTCTGAGCCACCAGACAGCACTGGGTAGTGCTTTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTG TCCTACAACGCCCTGGGAGCCCCTGCCCTGGCCAGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTG CACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAGGGTGATTCGGACCTCATGGAGCCTGTATTCCGATAC CTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTGACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTT AGAGACCTGTGCAGATGTCTCTCTCTGTGCCCCTCACTCATCTCACTGGATCTGTCTGCCAACCCTGAG ATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCCACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTT GGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTGGGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTC CGGGAACTGCAGCTGTGCAGCAGACGCCTCTGCGCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCC AGTCGGCCGGGCCCCGGCGAGTGCACGCTGGACCACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 11-TONSL N571A ATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGCAAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGG CGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGGGAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCT CTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGGGAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCC CACCGCAAGATCGGAGAGCGCCTGGCCGAGATGGAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAG TACCTGGAGCTGGCACATTCCCTGCGCAACCACACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGC ACCCACCTGGACATCTATGACCACTGCCAGTCGAGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAG AAGAGCTTGGCTATTGTGGATGAGGAGCTGGAGGGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGG ACCCGCCTCTATCTCAACCTGGGCCTCACCTTTGAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTAC TTCAGGAAGAGCATCTTCCTTGCGGAGCAGAACCACCTTTACGAGGACCTATTCCGCGCCCGCTACAAC CTGGGCACCATCCACTGGCGCGCGGGCCAGCACTCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAG TGTGCGCACACCATGAGGAAGCGGTTCATGGAGAGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAA GACCTGGGAGACTTTTTGGCTGCCAAGCGAGCCCTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCT GTGCAGAGGGCAGCCATCTGTCAGAACCTCCAGCATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTG GAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCCATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCC AAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCTTACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGAC AGACCGGGTGCTGAGCGGGCCATCATCCACGTGTCCCTGGCCACCACACTGGGAGACATGAAGGACCAC CATGGGGCCGTGCGCCACTATGAGGAGGAACTGAGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAG ACCTGGCTGAACATTGCACTGTCCCGCGAGGAGGCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTC CAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAGCGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTC CATACCGTGCAGCTGAGGCTGCAGCCCCAGGAGGCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGT GTAGCTGAAGATGAAGATGAGGAGGAGGAGGCGGAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTG GAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAGGACGACACCGATGGCCTGACCCCGCAGCTGGAGGAG GACGAGGAGCTTCAGGGCCACCTGGGCCGGCGGAAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGG GAGACCCTGCTGCACCGAGCCTGCATCGAGGGCCAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGC CACCCCCTTAACCCTCGGGACTACTGTGGCTGGACACCTCTGCACGAGGCCTGCGCCTACGGGCATCTA GAAATTGTCCGCTTCCTGCTGGACCACGGGGCCGCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGC ATCACCCCCCTCCACGATGCCCTCAACTGTGGCCACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGG GCGTCCGTCACCCTCCGCACTCGAAAGGGCCTCAGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTG TACCGCAGGGACCTGGACCTGGAGACGCGGCAGAAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCT GCCTCGGGCCAAGATCCCCACAGCTCCCAGGCCTTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAG ACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCCCCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAG GTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCACCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGG CCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAGGACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGG CCTCGGTGCTCGGCCACAGCACAACGGGTGGCAGCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCA GCCACAGCCAGCACCAGCCGGGCAGCCTACCAGGCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGG CTGGGGCCTGGCCCACCGCGGGGCCACAGCAAAGCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAG GAGTGCCTGGCTGGGGACTGGCTGGAGCTGGACATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGG GGCACTGGAGACAACCGCAGGCCCAGTAGTACCTCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCC CGAGCCAAGCAGGTCCGCCTGACCTGCATGCAGAGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGC CTGGCTTCAGAACCTCCAGGGAGCCCCAGCACCCCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCG GCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCTCCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTC TTCCTCATCCCTGTCCCACACAGCAGTGACACCCACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAG CGCTACTACCAGACCTGCGGGCTGCTGCCCAGGCTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCA CAGGACCTCATCCCTGATGTGCTGCAGAGCAATGACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTG CCCCCGTTGACTGACCGCTACCGCAGGGCCTGCCAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTG CAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCGTTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAG CTTACACCCCTGCTGCGGGCCCTCAAGCTGCACACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGG CTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCTGCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGAC CTCTCCTCCAATCACCTGGGTCCCGAAGGCCTGCGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACC TTGCAGAGTTTGGAGgaattagatctatcgatgaACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCC TCCCTCCTGCACGCCTGCCCCTTACTCAGCACCCTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTC TTTCTGAGCCACCAGACAGCACTGGGTAGTGCTTTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTG TCCTACAACGCCCTGGGAGCCCCTGCCCTGGCCAGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTG CACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAGGGTGATTCGGACCTCATGGAGCCTGTATTCCGATAC CTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTGACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTT AGAGACCTGTGCAGATGTCTCTCTCTGTGCCCCTCACTCATCTCACTGGATCTGTCTGCCAACCCTGAG ATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCCACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTT GGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTGGGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTC CGGGAACTGCAGCTGTGCAGCAGACGCCTCTGCGCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCC AGTCGGCCGGGCCCCGGCGAGTGCACGCTGGACCACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 12-TONSL N571A/GFP atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgta aacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaag ttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtg cagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggc tacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctg gggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggc atcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgcc ctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatc actctcggcatggacgagctgtacaagggcgcgccaATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGC AAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGGCGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGG GAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCTCTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGG GAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCCCACCGCAAGATCGGAGAGCGCCTGGCCGAGATG GAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAGTACCTGGAGCTGGCACATTCCCTGCGCAACCAC ACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGCACCCACCTGGACATCTATGACCACTGCCAGTCG AGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAGAAGAGCTTGGCTATTGTGGATGAGGAGCTGGAG GGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGGACCCGCCTCTATCTCAACCTGGGCCTCACCTTT GAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTACTTCAGGAAGAGCATCTTCCTTGCGGAGCAGAAC CACCTTTACGAGGACCTATTCCGCGCCCGCTACAACCTGGGCACCATCCACTGGCGCGCGGGCCAGCAC TCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAGTGTGCGCACACCATGAGGAAGCGGTTCATGGAG AGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAAGACCTGGGAGACTTTTTGGCTGCCAAGCGAGCC CTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGGCTGTGCAGAGGGCAGCCATCTGTCAGAACCTCCAG CATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTGGAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCC ATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCCAAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCT TACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGACAGACCGGGTGCTGAGCGGGCCATCATCCACGTG TCCCTGGCCACCACACTGGGAGACATGAAGGACCACCATGGGGCCGTGCGCCACTATGAGGAGGAACTG AGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAGACCTGGCTGAACATTGCACTGTCCCGCGAGGAG GCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTCCAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAG CGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTCCATACCGTGCAGCTGAGGCTGCAGCCCCAGGAG GCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGTGTAGCTGAAGATGAAGATGAGGAGGAGGAGGCG GAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTGGAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAG GACGACACCGATGGCCTGACCCCGCAGCTGGAGGAGGACGAGGAGCTTCAGGGCCACCTGGGCCGGCGG AAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGGGAGACCCTGCTGCACCGAGCCTGCATCGAGGGC CAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGCCACCCCCTTAACCCTCGGGACTACTGTGGCTGG ACACCTCTGCACGAGGCCTGCGCCTACGGGCATCTAGAAATTGTCCGCTTCCTGCTGGACCACGGGGCC GCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGCATCACCCCCCTCCACGATGCCCTCAACTGTGGC CACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGGGCGTCCGTCACCCTCCGCACTCGAAAGGGCCTC AGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTGTACCGCAGGGACCTGGACCTGGAGACGCGGCAG AAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCTGCCTCGGGCCAAGATCCCCACAGCTCCCAGGCC TTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAGACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCC CCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAGGTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCA CCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGGCCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAG GACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGGCCTCGGTGCTCGGCCACAGCACAACGGGTGGCA GCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCAGCCACAGCCAGCACCAGCCGGGCAGCCTACCAG GCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGGCTGGGGCCTGGCCCACCGCGGGGCCACAGCAAA GCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAGGAGTGCCTGGCTGGGGACTGGCTGGAGCTGGAC ATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGGGGCACTGGAGACAACCGCAGGCCCAGTAGTACC TCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCCCGAGCCAAGCAGGTCCGCCTGACCTGCATGCAG AGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGCCTGGCTTCAGAACCTCCAGGGAGCCCCAGCACC CCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCGGCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCT CCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTCTTCCTCATCCCTGTCCCACACAGCAGTGACACC CACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAGCGCTACTACCAGACCTGCGGGCTGCTGCCCAGG CTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCACAGGACCTCATCCCTGATGTGCTGCAGAGCAAT GACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTGCCCCCGTTGACTGACCGCTACCGCAGGGCCTGC CAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTGCAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCG TTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAGCTTACACCCCTGCTGCGGGCCCTCAAGCTGCAC ACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGGCTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCT GCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGACCTCTCCTCCAATCACCTGGGTCCCGAAGGCCTG CGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACCTTGCAGAGTTTGGAGgaattagatctatcgatg aACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCCTCCCTCCTGCACGCCTGCCCCTTACTCAGCACC CTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTCTTTCTGAGCCACCAGACAGCACTGGGTAGTGCT TTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTGTCCTACAACGCCCTGGGAGCCCCTGCCCTGGCC AGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTGCACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAG GGTGATTCGGACCTCATGGAGCCTGTATTCCGATACCTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTG ACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTTAGAGACCTGTGCAGATGTCTCTCTCTGTGCCCC TCACTCATCTCACTGGATCTGTCTGCCAACCCTGAGATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCC ACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTTGGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTG GGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTCCGGGAACTGCAGCTGTGCAGCAGACGCCTCTGC GCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCCAGTCGGCCGGGCCCCGGCGAGTGCACGCTGGAC CACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 13-TONSL D604A ATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGCAAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGG CGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGGGAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCT CTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGGGAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCC CACCGCAAGATCGGAGAGCGCCTGGCCGAGATGGAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAG TACCTGGAGCTGGCACATTCCCTGCGCAACCACACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGC ACCCACCTGGACATCTATGACCACTGCCAGTCGAGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAG AAGAGCTTGGCTATTGTGGATGAGGAGCTGGAGGGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGG ACCCGCCTCTATCTCAACCTGGGCCTCACCTTTGAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTAC TTCAGGAAGAGCATCTTCCTTGCGGAGCAGAACCACCTTTACGAGGACCTATTCCGCGCCCGCTACAAC CTGGGCACCATCCACTGGCGCGCGGGCCAGCACTCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAG TGTGCGCACACCATGAGGAAGCGGTTCATGGAGAGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAA GACCTGGGAGACTTTTTGGCTGCCAAGCGAGCCCTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCT GTGCAGAGGGCAGCCATCTGTCAGAACCTCCAGCATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTG GAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCCATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCC AAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCTTACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGAC AGACCGGGTGCTGAGCGGGCCATCATCCACGTGTCCCTGGCCACCACACTGGGAGACATGAAGGACCAC CATGGGGCCGTGCGCCACTATGAGGAGGAACTGAGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAG ACCTGGCTGAACATTGCACTGTCCCGCGAGGAGGCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTC CAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAGCGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTC CATACCGTGCAGCTGAGGCTGCAGCCCCAGGAGGCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGT GTAGCTGAAGATGAAGATGAGGAGGAGGAGGCGGAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTG GAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAGGACGACACCGATGGCCTGACCCCGCAGCTGGAGGAG GACGAGGAGCTTCAGGGCCACCTGGGCCGGCGGAAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGG GAGACCCTGCTGCACCGAGCCTGCATCGAGGGCCAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGC CACCCCCTTAACCCTCGGGACTACTGTGGCTGGACACCTCTGCACGAGGCCTGCAACTACGGGCATCTA GAAATTGTCCGCTTCCTGCTGGACCACGGGGCCGCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGC ATCACCCCCCTCCACGCTGCCCTCAACTGTGGCCACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGG GCGTCCGTCACCCTCCGCACTCGAAAGGGCCTCAGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTG TACCGCAGGGACCTGGACCTGGAGACGCGGCAGAAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCT GCCTCGGGCCAAGATCCCCACAGCTCCCAGGCCTTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAG ACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCCCCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAG GTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCACCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGG CCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAGGACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGG CCTCGGTGCTCGGCCACAGCACAACGGGTGGCAGCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCA GCCACAGCCAGCACCAGCCGGGCAGCCTACCAGGCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGG CTGGGGCCTGGCCCACCGCGGGGCCACAGCAAAGCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAG GAGTGCCTGGCTGGGGACTGGCTGGAGCTGGACATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGG GGCACTGGAGACAACCGCAGGCCCAGTAGTACCTCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCC CGAGCCAAGCAGGTCCGCCTGACCTGCATGCAGAGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGC CTGGCTTCAGAACCTCCAGGGAGCCCCAGCACCCCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCG GCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCTCCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTC TTCCTCATCCCTGTCCCACACAGCAGTGACACCCACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAG CGCTACTACCAGACCTGCGGGCTGCTGCCCAGGCTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCA CAGGACCTCATCCCTGATGTGCTGCAGAGCAATGACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTG CCCCCGTTGACTGACCGCTACCGCAGGGCCTGCCAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTG CAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCGTTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAG CTTACACCCCTGCTGCGGGCCCTCAAGCTGCACACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGG CTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCTGCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGAC CTCTCCTCCAATCACCTGGGTCCCGAAGGCCTGCGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACC TTGCAGAGTTTGGAGgaattagatctatcgatgaACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCC TCCCTCCTGCACGCCTGCCCCTTACTCAGCACCCTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTC TTTCTGAGCCACCAGACAGCACTGGGTAGTGCTTTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTG TCCTACAACGCCCTGGGAGCCCCTGCCCTGGCCAGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTG CACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAGGGTGATTCGGACCTCATGGAGCCTGTATTCCGATAC CTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTGACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTT AGAGACCTGTGCAGATGTCTCTCTCTGTGCCCCTCACTCATCTCACTGGATCTGTCTGCCAACCCTGAG ATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCCACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTT GGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTGGGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTC CGGGAACTGCAGCTGTGCAGCAGACGCCTCTGCGCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCC AGTCGGCCGGGCCCCGGCGAGTGCACGCTGGACCACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 14-TONSL D604A/GFP atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgta aacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaag ttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtg cagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggc tacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctg gggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggc atcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgcc ctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatc actctcggcatggacgagctgtacaagggcgcgccaATGAGCCTGGAGCGCGAGCTTCGCCAGCTGAGC AAGGCGAAAGCCAAGGCGCAGAGGGCCGGGCAGCGGCGCGAAGAGGCCGCGCTGTGCCACCAGCTGGGG GAGCTCCTGGCCGGCCATGGCCGCTACGCCGAGGCTCTGGAGCAGCACTGGCAGGAGCTGCAGCTTCGG GAGCGCGCTGACGACCCTCTGGGCTGTGCCGTGGCCCACCGCAAGATCGGAGAGCGCCTGGCCGAGATG GAGGACTACCCGGCTGCCTTGCAGCACCAGCACCAGTACCTGGAGCTGGCACATTCCCTGCGCAACCAC ACGGAGCTGCAGAGGGCCTGGGCCACCATCGGCCGCACCCACCTGGACATCTATGACCACTGCCAGTCG AGGGATGCTTTGCTGCAGGCACAGGCTGCCTTTGAGAAGAGCTTGGCTATTGTGGATGAGGAGCTGGAG GGGACACTGGCCCAGGGAGAGCTGAATGAGATGAGGACCCGCCTCTATCTCAACCTGGGCCTCACCTTT GAGAGCCTGCAGCAGACAGCCCTGTGCAACGATTACTTCAGGAAGAGCATCTTCCTTGCGGAGCAGAAC CACCTTTACGAGGACCTATTCCGCGCCCGCTACAACCTGGGCACCATCCACTGGCGCGCGGGCCAGCAC TCCCAGGCTATGCGCTGCTTGGAGGGTGCCCGGGAGTGTGCGCACACCATGAGGAAGCGGTTCATGGAG AGCGAGTGCTGCGTGGTTATTGCACAGGTCCTCCAAGACCTGGGAGACTTTTTGGCTGCCAAGCGAGCC CTGAAGAAGGCCTACAGGCTGGGCTCCCAGAAGCCTGTGCAGAGGGCAGCCATCTGTCAGAACCTCCAG CATGTGCTGGCAGTGGTCCGGCTGCAGCAACAGCTGGAAGAGGCTGAGGGCAGAGACCCTCAGGGTGCC ATGGTCATCTGTGAGCAGCTAGGGGACCTCTTCTCCAAGGCAGGAGACTTTCCCAGGGCAGCTGAGGCT TACCAGAAGCAGCTGCGTTTTGCTGAGCTGCTGGACAGACCGGGTGCTGAGCGGGCCATCATCCACGTG TCCCTGGCCACCACACTGGGAGACATGAAGGACCACCATGGGGCCGTGCGCCACTATGAGGAGGAACTG AGGCTGCGCAGCGGCAACGTGCTGGAGGAGGCCAAGACCTGGCTGAACATTGCACTGTCCCGCGAGGAG GCCGGCGATGCCTACGAGCTGCTGGCCCCGTGCTTCCAGAAAGCGCTCAGCTGTGCCCAGCAGGCCCAG CGTCCCCAGCTGCAGAGGCAGGTCTTGCAGCATCTCCATACCGTGCAGCTGAGGCTGCAGCCCCAGGAG GCCCCTGAGACCGAAACCAGACTGCGGGAGCTCAGTGTAGCTGAAGATGAAGATGAGGAGGAGGAGGCG GAGGAGGCGGCAGCCACAGCGGAGAGCGAAGCCCTGGAGGCCGGCGAGGTGGAGCTCTCAGAGGGCGAG GACGACACCGATGGCCTGACCCCGCAGCTGGAGGAGGACGAGGAGCTTCAGGGCCACCTGGGCCGGCGG AAGGGGAGCAAGTGGAACCGGCGAAACGACATGGGGGAGACCCTGCTGCACCGAGCCTGCATCGAGGGC CAGCTGCGCCGCGTCCAGGACCTTGTGAGGCAGGGCCACCCCCTTAACCCTCGGGACTACTGTGGCTGG ACACCTCTGCACGAGGCCTGCAACTACGGGCATCTAGAAATTGTCCGCTTCCTGCTGGACCACGGGGCC GCAGTGGACGACCCAGGTGGCCAGGGCTGCGAAGGCATCACCCCCCTCCACGCTGCCCTCAACTGTGGC CACTTCGAGGTGGCTGAGCTGCTGCTTGAACGGGGGGCGTCCGTCACCCTCCGCACTCGAAAGGGCCTC AGCCCGCTGGAGACGCTGCAGCAGTGGGTGAAGCTGTACCGCAGGGACCTGGACCTGGAGACGCGGCAG AAGGCCAGGGCCATGGAGATGCTGCTCCAGGCGGCTGCCTCGGGCCAAGATCCCCACAGCTCCCAGGCC TTCCACACCCCAAGCAGCCTTCTGTTTGACCCCGAGACCTCTCCTCCTTTGAGCCCCTGCCCAGAACCC CCCTCTAATAGCACTAGACTCCCAGAGGCCTCTCAGGTCCATGTCAGGGTCTCCCCAGGGCAGGCGGCA CCAGCCATGGCCAGGCCTCGGAGGAGCAGGCATGGGCCAGCCAGCAGCAGCAGCAGCTCAGAAGGCGAG GACAGCGCAGGCCCCGCACGGCCGTCCCAGAAGAGGCCTCGGTGCTCGGCCACAGCACAACGGGTGGCA GCCTGGACGCCTGGCCCCGCCAGCAACAGGGAAGCAGCCACAGCCAGCACCAGCCGGGCAGCCTACCAG GCAGCCATCCGGGGTGTGGGCAGTGCTCAGAGCCGGCTGGGGCCTGGCCCACCGCGGGGCCACAGCAAA GCCCTTGCCCCCCAGGCAGCGCTCATCCCGGAGGAGGAGTGCCTGGCTGGGGACTGGCTGGAGCTGGAC ATGCCCCTGACCCGCAGCCGCCGGCCCCGCCCCCGGGGCACTGGAGACAACCGCAGGCCCAGTAGTACC TCTGGGTCGGACAGTGAGGAGAGCAGGCCCCGTGCCCGAGCCAAGCAGGTCCGCCTGACCTGCATGCAG AGTTGCAGTGCGCCAGTTAACGCAGGGCCCAGCAGCCTGGCTTCAGAACCTCCAGGGAGCCCCAGCACC CCCAGGGTCTCAGAGCCCAGTGGGGACAGCTCTGCGGCAGGCCAGCCCTTGGGTCCGGCCCCGCCCCCT CCCATCCGGGTTCGAGTTCAAGTTCAGGATCATCTCTTCCTCATCCCTGTCCCACACAGCAGTGACACC CACTCTGTGGCCTGGCTGGCCGAGCAGGCGGCCCAGCGCTACTACCAGACCTGCGGGCTGCTGCCCAGG CTCACCCTACGGAAAGAGGGGGCCCTGCTGGCCCCACAGGACCTCATCCCTGATGTGCTGCAGAGCAAT GACGAGGTGTTGGCTGAGGTGACTTCGTGGGACCTGCCCCCGTTGACTGACCGCTACCGCAGGGCCTGC CAGAGCCTGGGGCAAGGGGAGCACCAACAGGTGCTGCAGGCCGTGGAGCTCCAGGGCTTGGGCCTCTCG TTCAGCGCCTGCTCCCTGGCCCTGGACCAGGCCCAGCTTACACCCCTGCTGCGGGCCCTCAAGCTGCAC ACAGCACTCCGGGAGCTGCGCCTGGCAGGGAACCGGCTGGGGGACAAGTGTGTGGCTGAGCTGGTGGCT GCCCTGGGCACCATGCCCAGCCTGGCCCTCCTTGACCTCTCCTCCAATCACCTGGGTCCCGAAGGCCTG CGCCAGCTTGCCATGGGGCTCCCAGGCCAAGCCACCTTGCAGAGTTTGGAGgaattagatctatcgatg aACCCCCTGGGGGACGGCTGTGGCCAGTCCCTGGCCTCCCTCCTGCACGCCTGCCCCTTACTCAGCACC CTGCGCCTGCAGGCGTGTGGCTTCGGCCCCAGCTTCTTTCTGAGCCACCAGACAGCACTGGGTAGTGCT TTCCAAGATGCTGAGCACCTGAAGACCCTGTCCCTGTCCTACAACGCCCTGGGAGCCCCTGCCCTGGCC AGGACCCTGCAGAGCCTGCCCGCCGGCACCCTCCTGCACTTAGAGCTCAGCTCCGTGGCAGCCGGCAAG GGTGATTCGGACCTCATGGAGCCTGTATTCCGATACCTGGCCAAGGAAGGCTGTGCTCTAGCCCACCTG ACCCTGTCTGCAAACCACCTGGGGGACAAGGCTGTTAGAGACCTGTGCAGATGTCTCTCTCTGTGCCCC TCACTCATCTCACTGGATCTGTCTGCCAACCCTGAGATCAGCTGTGCCAGCTTGGAAGAGCTCCTGTCC ACCCTCCAAAAGCGGCCCCAAGGCCTTAGCTTCCTTGGCCTGTCAGGCTGCGCCGTCCAGGGTCCCCTG GGCCTGGGCCTGTGGGACAAGATAGCCGCGCAGCTCCGGGAACTGCAGCTGTGCAGCAGACGCCTCTGC GCTGAGGACAGGGACGCCCTGCGCCAGCTGCAGCCCAGTCGGCCGGGCCCCGGCGAGTGCACGCTGGAC CACGGCTCCAAGCTCTTCTTTCGGCGCCTCTAG SEQ ID NO: 15-TONSL FUSION (MCM2 HBD-TONSL ARD) GGATCCGGGCCCCTGGAGGAAGAAGAGGATGGAGAGGAGCTCATTGGAGATGGCATGGAAAGGGACTAC CGCGCCATCCCAGAGCTGGACGCCTATGAGGCCGAGGGACTGGCTCTGGATGATGAGGACGTAGAGGAG CTGACGGCCAGTCAGAGGGAGGCAGCAGAGCGGGCCATGCGGCAGCGTGACCGGGAGGCTGGCCGGGGC CTGGGCCGAGGAGGTGGAGGTGGCCACCTGGGCCGGCGGAAGGGGAGCAAGTGGAACCGGCGAAACGAC ATGGGGGAGACCCTGCTGCACCGAGCCTGCATCGAGGGCCAGCTGCGCCGCGTCCAGGACCTTGTGAGG CAGGGCCACCCCCTTAACCCTCGGGACTACTGTGGCTGGACACCTCTGCACGAGGCCTGCAACTACGGG CATCTAGAAATTGTCCGCTTCCTGCTGGACCACGGGGCCGCAGTGGACGACCCAGGTGGCCAGGGCTGC GAAGGCATCACCCCCCTCCACGATGCCCTCAACTGTGGCCACTTCGAGGTGGCTGAGCTGCTGCTTGAA CGGGGGGCGTCCGTCACCCTCCGCACTCGAAAGGGCCTCAGCCCGCTGGAGACGCTGCAGCAGTGGGTG AAGCTGTACCGCAGGGACCTGGACCTGGAGACGCGGCAGAAGGCCAGGGCCATGGAGATGCTGCTCCAG GCGGCTGCCTCGGGCCAAGATCCCCACAGCTCCCAGGCCTTCCACACCCCAAGCAGCCTTCTGTTTGAC CCCGAGACCTCTTAACTCGAG SEQ ID NO: 16-TONSL wild-type (amino acids mutated in single-mutants are highlighted) >gi|187608777|ref|NP_038640.1| tonsoku-like protein [Homo sapiens] MSLERELRQLSKAKAKAQRAGQRREEAALCHQLGELLAGHGRYAEALEQHWQELQLRERADDPLGCAVA HRKIGERLAEMEDYPAALQHQHQYLELAHSLRNHTELQRAWATIGRTHLDIYDHCQSRDALLQAQAAFE KSLAIVDEELEGTLAQGELNEMRTRLYLNLGLTFESLQQTALCNDYFRKSIFLAEQNHLYEDLFRARYN LGTIHWRAGQHSQAMRCLEGARECAHTMRKRFMESECCVVIAQVLQDLGDFLAAKRALKKAYRLGSQKP VQRAAICQNLQHVLAVVRLQQQLEEAEGRDPQGAMVICEQLGDLFSKAGDFPRAAEAYQKQLRFAELLD RPGAERAIIHVSLATTLGDMKDHHGAVRHYEEELRLRSGNVLEEAKTWLNIALSREEAGDAYELLAPCF QKALSCAQQAQRPQLQRQVLQHLHTVQLRLQPQEAPETETRLRELSVAEDEDEEEEAEEAAATAESEAL EAGEVELSEGEDDTDGLTPQLEEDEELQGHLGRRKGSKWNRRNDMG E TTLLHRACIEGQLRRVQDLVRQ GHPLNPR D YCG W TPLH E AC N YGHLEIVRFLLDHGAAVDDPGGQGCEGITPLH D ALNCGHFEVAELLLER GASVTLRTRKGLSPLETLQQWVKLYRRDLDLETRQKARAMEMLLQAAASGQDPHSSQAFHTPSSLLFDP ETSPPLSPCPEPPSNSTRLPEASQAHVRVSPGQAAPAMARPRRSRHGPASSSSSSEGEDSAGPARPSQK RPRCSATAQRVAAWTPGPASNREAATASTSRAAYQAAIRGVGSAQSRLGPGPPRGHSKALAPQAALIPE EECLAGDWLWLDMPLTRSRRPRPRGTGDNRRPSSTSGSDSEESRPRARAKQVRLTCMQSCSAPVNAGPS SLASEPPGSPSTPRVSEPSGDSSAAGQPLGPAPPPPIRVRVQVQDHLFLIPVPHSSDTHSVAWLAEQAA QRYYQTCGLLPRLTLRKEGALLAPQDLIPDVLQSNDEVLAEVTSWDLPPLTDRYRRACQSLGQGEHQQV LQAVELQGLGLSFSACSLALDQAQLTPLLRALKLHTALRELRLAGNRLGDKCVAELVAALGTMPSLALL DLSSNHLGPEGLRQLAMGLPGQATLQSLEELDLSMNPLGDGCGQSLASLLHACPLLSTLRLQACQFGPS FFLSHQTALGSAFQDAEHLKTLSLSYNALGAPALARTLQSLPAGTLLHLELSSVAAGKGDSDLMEPVFR YLAKEGCALAHTLSANHLGDKAVRDLCRCLSLCPSLISLDLSANPEISCASLEELLSTLQKRPQGLSFL GLSGCAVQGPLGLGWDKIAAQLRELQLCSRRLCAEDRDALRQLQPSRPGPGECTLDHGSKLFFRRL SEQ ID NO: 17-Protein sequences of TONSL E530A single mutants (mutated alanins are highlighted) MSLERELRQLSKAKAKAQRAGQRREEAALCHQLGELLAGHGRYAEALEQHWQELQLRERADDPLGCAVA HRKIGERLAEMEDYPAALQHQHQYLELAHSLRNHTELQRAWATIGRTHLDIYDHCQSRDALLQAQAAFE KSLAIVDEELEGTLAQGELNEMRTRLYLNLGLTFESLQQTALCNDYFRKSIFLAEQNHLYEDLFRARYN LGTIHWRAGQHSQAMRCLEGARECAHTMRKRFMESECCVVIAQVLQDLGDFLAAKRALKKAYRLGSQKP VQRAAICQNLQHVLAVVRLQQQLEEAEGRDPQGAMVICEQLGDLFSKADFPRAAEAYQKQLRFAELLDR PGAERAIIHVSLATTLGDMKDHHFAVRHYEEELRLRSGNVLEEAKTWLNIALSREEAGDAYELLAPCFQ KALSCAQQAQRPQLQRQVLQHLHTVQLRLQPQEAPETETRLRELSVAEDEDEEEEAEEAAATAESEALE AGEVELSEGEDDTDGLTPQLEEDEELQGHLGRRKGSKWNRRNDMGATLLHRACIEGQLRRVQDLVRQGH PLNPRDYCGWTPLHEACNYGHLEIVRFLLDHGAAVDDPGGQGCEGITPLHDALNCGHFEVAELLERGAS VTLRTRKGLSPLETLQQWVKLYRRDLDLETRQKARAMEMLLQAAASGQDPHSSQAFHTPSSLLFDPETS PPLSPCPEPPSNSTRLPEASQAHVRVSPGQAAPAMARPRRSRHGPASSSSSSEGEDSAGPARPSQKRPR CSATAQRVAAWTPGPASNREAATASTSRAAYQAAIRGVGSAQSRLGPGPPRGHSKALAPQAALIPEEEC LAGDWLELDMPLTRSRRPRPRGTGDNRRPSSTSGSDSEESRPRARAKQVRLTCMQSCSAPVNAGPSSLA SEPPGSPSTPRVSEPSGDSSAAGQPLGPAPPPPIRVRVQVQDHLFLIPVPHSSDTHSVAWLAEQAAQRY YQTCGLLPRLTLRKEGALLAPQDLIPDVLQSNDEVLAEVTSWDLPPLTDRYRRACQSLGQGEHQQVLQA VELQGLGLSFSACSLALDQAQLTPLLRALKLHTALRELRLAGNRLGDKCVAELVAALGTMPSLALLDLS SNHLGPEGLRQLAMGLPGQATLQSLEELDLSMNPLGDGCGQSLASLLHACPLLSTLRLQACGFGPSFFL SHQTALGSAFQDAEHLKTLSLSYNALGAPALARTLQSLPAGTLLHLELSSVAAGKGDSDLMEPVFRYLA KEGCALAHLTLSANHLGDKAVRDLCRCLSLCPSLISLDLSANPEISCASLEELLSTLQKRPQGLSFLGL SGCAVQGPLGLGLWDKIAAQLRELQLCSRRLCAEDRDALRQLQPSRPGPGECTLDHGSKLFFRRL SEQ ID NO: 18-Protein sequences of TONSL D559A MSLERELRQLSKAKAKAQRAGQRREEAALCHQLGELLAGHGRYAEALEQHWQELQLRERADDPLGCAVA HRKIGERLAEMEDYPAALQHQHQYLELAHSLRNHTELQRAWATIGRTHLDIYDHCQSRDALLQAQAAFE KSLAIVDEELEGTLAQGELNEMRTRLYLNLGLTFESLQQTALCNDYFRKSIFLAEQNHLYEDLFRARYN LGTIHWRAGQHSQAMRCLEGARECAHTMRKRFMESECCVVIAQVLQDLGDFLAAKRALKKAYRLGSQKP VQRAAICQNLQHVLAVVRLQQQLEEAEGRDPQGAMVICEQLGDLFSKAGDFPRAAEAYQKQLRFAELLD RPGAERAIIHVSLATTLGDMKDHHGAVRHYEEELRLRSGNVLEEAKTWLNIALSREEAGDAYELLAPCF QKALSCAQQAQRPQLQRQVLQHLHTVQLRLQPQEAPETETRLRELSVAEDEDEEEEAEEAAATAESEAL EAGEVELSEGEDDTDGLTPQLEEDEELQGHLGRRKGSKWNRRNDMGETLLHRACIEGQLRRVQDLVRQG HPLNPRAYCGWTPLHEACNYGHLEIVRFLLDHGAAVDDPGGQGCEGITPLHDALNCGHFEVAELLERGA SVTLRTRKGLSPLETLQQWVKLYRRDLDLETRQKARAMEMLLQAAASGQDPHSSQAFHTPSSLLFDPET SPPLSPCPEPPSNSTRLPEASQAHVRVSPGQAAPAMARPRRSRHGPASSSSSSEGEDSAGPARPSQKRP RCSATAQRVAAWTPGPASNREAATASTSRAAYQAAIRGVGSAQSRLGPGPPRGHSKALAPQAALIPEEE CLAGDWLELDMPLTRSRRPRPRGTGDNRRPSSTSGSDSEESRPRARAKQVRLTCMQSCSAPVNAGPSSL ASEPPGSPSTPRVSEPSGDSSAAGQPLGPAPPPPIRVRVQVQDHLFLIPVPHSSDTHSVAWLAEQAAQR YYQTCGLLPRLTLRKEGALLAPQDLIPDVLQSNDEVLAEVTSWDLPPLTDRYRRACQSLGQGEHQQVLQ QVELQGLGLSFSACSLALDQAQLTPLLRALKLHTALRELRLAGNRLGDKCVAELVAALGTMPSLALLDL SSNHLGPEGLRQLAMGLPGQATLQSLEELDLSMNPLGDGCGQSLASLLHACPLLSTLRLQACGFGPSFF LSHQTALGSAFQDAEHLKTLSLSYNALGAPALARTLQSLPAGTLLHLELSSVAAGKGDSDLMEPVFRYL AKEGCALAHLTLSANHLGDKAVRDLCRCLSLCPSLISLDLSANPEISCASLEELLSTLQKRPQGLSFLG LSGCAVQGPLGLGLWDKIAAQLRELQLCSRRLCAEDRDALRQLQPSRPGPGECTLDHGSKLFFRRL SEQ ID NO: 19-Protein sequences of TONSL W563A MSLERELRQLSKAKAKKAQRAGQRREEAALCHQLGELLAGHGRYAEALEQHWQELALRERADDPLGCAV HRKIGERLAEMEDYPAALQHQHQYLELAHSLRNHTELQRAWATIGRTHLDIYDHCQSRDALLQAQAAFE KSLAIVDEELEGTLAQGELNEMRTRLYLNLGLTFESLQQTALCNDYFRKSIFLAEQNHLYEDLFRARYN LGTIHWRAGQHSQAMRCLEGARECAHTMRKRFMESECCVVIAQVLQDLGDFLAAKRALKKAYRLGSQKP VQRAAICQNLQHVLAVVRLQQQLEEAEGRDPQGAMVICEQLGDLFSKAGDFPRAAEAYQKQLRFAELLD RPGAERAIIHVSLATTLGDMKDHHGAVRHYEEELRLRSGNVLEEAKTWLNIALSREEAGDAYELLAPCF QKALSCAQQAQRPQLQRQVLQHLHTVQLRLQPQEAPETETRLRELSVAEDEDEEEEAEEAAATAESEAL EAGEVELSEGEDDTDGLTPQLEEDEELQGHLGRRKGSKWNRRNDMGETLLHRACIEGQLRRVQDLVRQG HPLNPRDYCGATPLHEACNYGHLEIVRFLLDHGAAVDDPGGQGCEGITPLHDALNCGHFEVAELLERGA SVTLRTRKGLSPLETLQQWVKLYRRDLDLETRQKARAMEMLLQAAASGQDPHSSQAFHTPSSLLFDPET SPPLSPCPEPPSNSTRLPEASQAHVRVSPGQAAPAMARPRRSRHGPASSSSSSEGEDSAGPARPSQKRP RCSATAQRVAAWTPGPASNREAATASTSRAAYQAAIRGVGSAQRSLGPGPPRGHSKALAPQAALIPEEE CLAGDWLELDMPLTRSRRPRPRGTGDNRRPSSTSGSDSEESRPRARAHQVRLTCMQSCSAPVNAGPSSL ASEPPGSPSTPRVSEPSGDSSAAGQPLGPAPPPPIRVRVQVQDHLFLIPVPHSSDTHSVAWLAEQAAQR YYQTCGLLPRLTLRKEGALLAPQDLIPDVLQSNDEVLAEVTSWDLPPLTDRYRRACQSLGQGEHQQVLQ AVELQGLGLSFSACSLALDQAQLTPLLRALKLHTALRELRLAGNRLGDKCVAELVAALGTMPSLALLDL SSNHLGPEGLRQLAMGLPGQATLQSLEELDLSMNPLGDGCGQSLASLLHACPLLSTLRLQACGFGPSFF LSHQTALGSAFQDAEHLKTLSLSYNALGAPALARTLQSLPAGTLLHLELSSVAAGKGDSDLMEPVFRYL AKEGCALAHLTLSANHLGDKAVRDLCRCLSLCPSLISLDLSANPEISCASLEELLSTLQKRPQGLSFLG LSGCAVQGPLGLGLWDKIAAQLRELQLCSRRLCAEDRDALRQLQPSRPGPGECTLDHGSKLFFRRL SEQ ID NO: 20-Protein sequences of TONSL E568A MSLERELRQLSKAKAKAQRAGQRREEAALCHQLGELLAGHGRYAEALEQHWQELQLRERADDPLGCAVA HRKIGERLAEMEDYPAALQHQHQYLELAHSLRNHTELQRAWATIGRTHLDIYDHCQSRDALLQAQAAFE KSLAIVDEELEGTLAQGELNEMRTRLYLNLGLTFESLQQTALCNDYFRKSIFLAEQNHLYEDLFRARYN LGTIHWRAGQHSQAMRCLEGARECAHTMRKRFMESECCVVIAQVLQDLGDFLAAKRALKKAYRLGSQKP VQRAAICQNLQHVLAVVRLQQQLEEAEGRDPQGAMVICEQLGDLFSKAGDFPRAAEAYQKQLRFAELLD RPGAERAIIHVSLATTLGDMKDHHGAVRHYEEELRLRSGNVLEEAKTWLNIALSREEAGDAYELLAPCF QKALSCAQQAQRPQLQRQVLQHLHTVQLRLQPQEAPETETRLRELSVAEDEDEEEEAEEAAATAESEAL EAGEVELSEGEDDTDGLTPQLEEDEELQGHLGRRKGSKWNRRNDMGETLLHRACIEGQLRRVQDLVRQG HPLNPRDYCGWTPLHAACNYGHLEIVRFLLDHGAAVDDPGGQGCEGTIPLHDALNCGHFEVAELLLERG ASVTLRTRKGLSPLETLQQWVKLYRRDLDLETRQKARAMEMLLQAAASGQDPHSSQAFHTPSSLLFDPE TSPPLSPCPEPPSNSTRLPEASQAHVRVSPGQAAPAMARPRRSRHGPASSSSSSEGEDSAGPARPSQKR PRCSATAQRVAAWTPGPASNREAATASTSRAAYQAAIRGVGSAQSRLGPGPPRGHSKALAPQAALIPEE ECLAGDWLELDMPLTRSRRPRPRGTGDNRRPSSTSGSDSEESRPRARAKQVRLTCMQSCSAPVNAGPSS LASEPPGSPSTPRVSEPSGDSSAAGQPLGPAPPPPIRVRVQVQDHLFLIPVPHSSDTHSCAWLAEQAAQ RYYQTCGLLPRLTLRKEGALLAPQDLIPDVLQSNDEVLAEVTSWDLPPLTDRYRRACQSLGQGEHQQVL QAVELQGLGLSFSACSLALDQAQLTPLLRALKLHTALRELRLAGNRLGDKCVAELVAALGTMPSLALLD LSSNHLGPEGLRQLAMGLPGQATLQSLEELDLSMNPLGDGCGQSLASLLHACPLLSTLRLQACGFGPSF FLSHQTALGSAFQDAEHLKTLSLSYNALGAPALARTLQSLPAGTLLHLELSSVAAGKGDSDLMEPVFRY LAKEGCALAHLTLSANHLGDKAVRDLCRCLSLCPSLISLDLSANPEISCASLEELLSTLQKRPQGLSFL GLSGCAVQGPLGLGLWDKIAAQLRELQLCSRRLCAEDRDALRQLQPSRPGPGECTLDHGSKLFFRRL SEQ ID NO: 21-Protein sequences of TONSL N571A MSLERELRQLSKAKAKAQRAGQRREEAALCHQLGELLAGHGRYAEALEQHWQELQLRERADDPLGCAVA HRKIGERLAEMEDYPAALQHQHQYLELAHSLRNHTELQRAWATIGRTHLDIYDHCQSRDALLQAQAAFE KSLAIVDEELEGTLAQGELNEMRTRLYLNLGLTFESLQQTALCNDYFRKSIFLAEQNHLYEDLFRARYN LGTIHWRAGQHSQAMRCLEGARECAHTMRKRFMESECCVVIAQVLQDLGDFLAAKRALKKAYRLGSQKP VQRAAICQNLQHVLAVVRLQQQLEEAEGRDPQGAMVICEQLGDLFSKAGDFPRAAEAYQKQLRFAELLD RPGAERAIIHVSLATTLGDMKDHHGAVRHYEEELRLRSGNVLEEAKTWLNIALSREEAGDAYELLAPCF QKALSCAQQAQRPQLQRQVLQHLHTVQLRLQPQEAPETETRLRELSVAEDEDEEEEAEEAAATAESEAL EAGEVELSEGEDDTDGLTPQLEEDEELQGHLGRRKGSKWNRRNDMGETLLHRACIEGQLRRVQDLVRQG HPLNPRDYCGWTPLHEACAYGHLEIVRFLLDHGAAVDDPGGQGCEGITPLHDALNCGHFEVAELLLERG ASVTLRTRKGLSPLETLQQWVKLYRRDLDLETRQKARAMEMLLQAAASGQDPHSSQAFHTPSSLLFDPE TSPPLSPCPEPPSNSTRLPEASQAHVTVSPGQAAPAMARPRRSRHGPASSSSSSEGEDSAGPARPSQKR PRCSATAQRVAAWTPGPASNREAATASTSRAAYQAAIRGVGSAQSRLGPGPPRGHSKALAPQAALIPEE ECLAGDWLELDMPLTRSRRPRPRGTGDNRRPSSTSGSDSEESRPRARAKQVRLTCMQSCSAPVNAGPSS LASEPPGSPSTPRVSEPSGDSSAAGQPLGPAPPPPIRVRVQVQDHLFLIPVPHSSDTHSVAWLAEQAAQ RYYQTCGLLPRLTLRKEGALLAPQDLIPDVLQSNDEVLAEVTSWDLPPLTDRYRRACQSLGQGEHQQVL QAVELQGLGLSFSACSLALDQAQLTPLLRALKLHTALRELRLAGNRLGDKCVAELVAALGTMPSLALLD LSSNHLGPEGLRQLAMGLPGQATLQSLEELDLSMNPLGDGCGQSLASLLHACPLLSTLRLQACGFGPSF FLSHQTALGSAFQDAEHLKTLSLSYNALGAPALARTLQSLPAGTLLHLELSSVAAGKGDSDLMEPVFRY LAKEGCALAHLTLSANHLGDKAVRDLCRCLSLCPSLISLDLSANPEISCASLEELLSTLQKRPQGLSFL GLSGCAVQGPLGLGLWDKIAAQLRELQLCSRRLCAEDRDALRQLQPSRPGPGECTLDHGSKLFFRRL SEQ ID NO: 22-Protein sequences of TONSL D604A MSLERELRQLSKAKAKAQRAGQRREEAALCHQLGELLAGHGRYAEALEQHWQELQLRERADDPLGCAVA HRKIGERLAEMEDYPAALQHQHGYLELAHSLRNHTELQRAWATIGRTHLDIYDHCQSRDALLQAQAAFE KSLAIVDEELEGTLAQGELNEMRTRLYLNLGLTFESLQQTALCNDYFRKSIFLAEQNHLYEDLFRARYN LGTIHWRAGQHSQAMRCLEGARECAHTMRKRFMESECCVVIAQVLQDLGDFLAAKRALKKAYRLGSQKP VQRAAICQNLQHVLAVVRLQQQLEEAEGRDPQGAMVICEQLGDLFSKAGDFPRAAEAYQKQLRFAELLD RPGAERAIIHVSLATTLGDMKDHHGAVRHYEEELRLRSGNVLEEAKTWLNIALSREEAGDAYELLAPCF QKALSCAQQAQRPQLQRQVLQHLHTVQLRLQPQEAPETETRLRELSVAEDEDEEEEAEEAAATAESEAL EAGEVELSEGEDDTDGLTPQLEEDEELQGHLGRRKGSKWNRRNDMGETLLHRACIEGQLRRVQDLVRQG HPLNPRDYCGWTPLHEACNYGHLEIVRFLLDFGAAVDDPGGQGCEGITPLHAALNCGHFEVAELLLERG ASVTLRTRKGLSPLETLQQWVKLYRRDLDLETRQKARAMELLQAAASGQDPHSSQAFHTPSSLLFDPET SPPLSPCPEPPSNSTRLPEASQAHVRVSPGQAAPAMARPRRSRHGPASSSSSSEGEDSAGPARPSQKRP RCSATAQRVAAWTPGPASNREAATASTSRAAYQAAIRGVGSAQSRLGPGPPRGHSKALAPQAALIPEEE CLAGDWLELDMPLTRSRRPRPRGTGDNRRPSSTSGSDSEESRPRARAKQVRLTCMQSCSAPVNAGPSSL ASEPPGSPSTPRVSEPSGDSSAAGQPLGPAPPPPIRVRVQVQDHLFLIPVPHSSDTHSVAWLAEQAAQR YYQTCGLLPRLTLRKEGALLAPQDLIPDVLQSNDEVLAEVTSWDLPPLTDRYRRACQSLGQGEHQQVLQ AVELQGLGLSFSACSLALDQAQLTPLLRALKLHTALRELRLANGNRLGDKCVAELVAALGTMPSLALLD LSSNHLGPEGLRQLAMGLPGQATLQSLEELDLSMNPLGDGCGQSLASLLHACPLLSTLRLQACGFGPSF FLSHQTALGSAFQDAEHLKTLSLSYNALGAPALARTLQSLPAGTLLHLELSSVAAGKGDSDLMEPVFRY LAKEGCALAHLTLSANHLGDKAVRDLCRCLSLCPSLISLDLSANPEISCASLEELLSTLQKRPQGLSFL GLSGCAVQGPLGLGLWDKIAAQLRELQLCSRRLCAEDRDALRQLQPSRPGPGECTLDHGSKLFFRRL SEQ ID NO: 23-protein sequence of Histone H4 >sp|P62805|H4_HUMAN Histone H4 OS = Homo sapiens MSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRD AVTYTEHAKRKTVTAMDVVYALKRQGRTLYGFGG SEQ ID NO: 24-protein sequence of MCM2 >sp|P49736|MCM2_HUMAN DNA replication licensing factor MCM2 OS = Homo sapiens MAESSESFTMASSPAQRRRGNDPLTSSPGRSSRRTDALTSSPGRDLPPFEDESEGLLGTEGPLEEEEDG EELIGDGMERDYRAIPELDAYEAEGLALDDEDVEELTASQREAAERAMRQRDREAGRGLGRMRRGLLYD SDEEDEERPARKRRQVERATEDGEEDEEMIESIENLEDLKGHSVREWVSMAGPRLEIHHRFKNFLRTHV DSHGHNVFKERISDMCKENRESLVVNYEDLAAREHVLAYFLPEAPAELLQIFDEAALEVVLAMYPKYDR ITNHIHVRISHLPLVEELRSLRQLHLNQLIRTSGVVTSCTGVLPQLSMVKYNCNKCNVFLGPFCQSQNQ EVKPGSCPECQSAGPFEVNMEETIYQNYQRIRIQESPGKVAAGRLPRSKDAILLADLVDSCKPGDEIEL TGIYHNNYDGSLNTANGFPVFATVILANHVAKKDNKVAVGELTDEDVKMITSLSKDQQIGEKIFASIAP SIYGHEDIKRGLALALFGGEPKNPGGKHKVRGDINVLLCGDPGTAKSQFLKYIEKVSSRAIFTTGQGAS AVGLTAYVQRHPVSREWTLEAGALVLADRGVCLIDEFDKMNDQDRTSIHEAMEQQSISISKAGIVTSLQ ARCTVIAAANPIGGRYDPSLTFSENVDLTEPIISRFDILCVVRDTVDPVQDEMLARFVVGSHVRHHPSN KEEGLANGSAAEPAMPNTYGVEPLPQEVLKKYIIYAKERVHPKLNQMDQDKVAKMYSDLRKESMATGSI PITVRHIESMIRMAEAHARIHLRDYVIEDDVNMAIRVMLESFIDTQKFSVMRSMRKTFARYLSFRRDNN ELLLFILKQLVAEQVTYQRNRFGAQQDTIEVPEKDLVDKARQINIHNLSAFYDSELFFRMNKFSHDLKR KMILQQF SEQ ID NO: 25-protein sequence of Histone H3.3 >sp|P84243|H33_HUMAN Histone H3.3 OS = Homo sapiens MARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQ RLVREIAQDFKTDLRFQSAAIGALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA SEQ ID NO: 26 RHXK SEQ ID NO: 27 RHXKVL SEQ ID NO: 28 RHXKVLR SEQ ID NO: 29 Ala-Lys-Arg-His-Arg-Lys-Val-Leu-Arg SEQ ID NO: 30 Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-Leu-Arg SEQ ID NO: 31 Lys-Gly-Gly-Ala-Lys-Arg-His-Ala-Lys-Val-Leu-Arg SEQ ID NO: 32 Lys-Gly-Gly-Ala-Ala-Arg-His-Arg-Lys-Val-Leu-Arg SEQ ID NO: 33 Leu-Gly-Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-Leu-Arg-Asp-Asn-Ile SEQ ID NO: 34-protein sequence of Histone H4 SGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRDA VTYTEHAKRKTVTAMDVVYALKRQGRTLYGFGG

ITEMS OF THE INVENTION

Some aspects of the invention may further be identified by the following items:

1. A small molecule, which targets the conformational space of the TONSL ARD occupied by the histone H4 tail encompassing residues K12-R23 and acting to prevent or disrupt the binding of the H4 tail K12-R23 with the TONSL ARD via direct competition or via allosteric disruption of the binding pocket.

2. A small molecule according to item 1, that targets the H4 tail spanning residues Lys12 to Arg23 through intermolecular hydrogen-bonding, electrostatic and/or van der Waals interactions.

3. A small molecule according to any of items 1-2, wherein the molecule targets the intermolecular contacts spanning the Lys12-Gly13-Gly14-Ala15 segment of H4.

4. A small molecule according to any of items 1-3, wherein the molecule targets the hydrophobic interactions between residues Gly13, Gly14 and Ala15 of H4 and residues Asn507, Cys508, Trp641, Tyr645 and Leu649 of ARD.

5. A small molecule according to any of items 1-4, wherein the molecule targets the hydrogen bonds between the main-chain O of H4 Gly14 and Nε1 of ARD Trp641, and between the main-chain N of H4 Ala15 and Oη of ARD Tyr645.

6. A small molecule according to any of items 1-5, wherein the molecule targets the main-chain O of H4 Lys16 hydrogen bonds with the Nδ2 of ARD Asn571.

7. A small molecule according to any of items 1-6, wherein the molecule targets the side-chain of H4 Arg17, which stacks over the side-chains of ARD Tyr572 and Cys608, while its Nη1 atom forms two hydrogen bonds with main-chain O and Oδ1 of ARD Asn571.

8. A small molecule according to any of items 1-7, wherein the molecule targets the side-chain of H4 H18, which penetrates into a pocket lined by four strictly conserved residues (Trp563, Glu568, Asn571 and Asp604) and is positioned over His567 of ARD.

9. A small molecule according to any of items 1-8, wherein the molecule targets the side chain of H4 His18, which is stacked between Trp563 and Asn571 and forms hydrogen bonds to Glu568 and Asp604 of ARD.

10. A small molecule according to any of items 1-9, wherein the molecule targets the main-chain O of H4 Arg19, which forms a hydrogen bond with Nε1 of Trp563 and its side-chain forms contacts with Cys561 and Gly595 of ARD.

11. A small molecule according to any of items 1-10, wherein the molecule targets the H4 Lys20 residue thst is bound within an acidic surface pocket on ARD adjacent to the H4 His18 binding pocket.

12. A small molecule according to any of items 1-11, wherein the molecule targets side-chain of H4 Lys20 which interacts with the side-chain of Met528 and contacts the edge of Trp563 of ARD, while the main-chain atoms of H4 Lys20 packs against Cys561 of ARD.

13. A small molecule according to any of items 1-12, wherein the molecule targets the Nδ atom of H4 Lys20 that forms three strong hydrogen bonds (distance <3 Å) with the side-chains of strictly conserved residues Glu530, Asp559 and Glu568 of ARD, which surround H4 Lys20 within a regular triangle-like alignment.

14. A small molecule according to any of items 1-13, wherein the molecule targets intermolecular contacts spanning the Val21-Leu22-Arg23 segment of H4, which includes contacts between side-chains of H4 Val21 with Tyr560 and Cys561 of ARD, while H4 Leu22 interacts with Asp527 and Met528 of ARD.

15. A small molecule according to any of items 1-14, wherein the molecule targets the main-chain N of H4 Arg23 which forms a hydrogen bond with the main-chain O of Asp527 of ARD, while the side-chain packs against the side-chain of Tyr560 of ARD.

16. A small molecule according to any of items 1-15, capable of blocking histone reader domains in a protein selected from the group consisting of TONSL, BARD1 and ANKRD11.

17. A small molecule according to any of items 1-16 for use as a medicament.

18. A small molecule according to any of items 1-17 for use in treatment of cancer.

19. A method of selecting or designing a small molecule capable of interfering with the histone H4H18 and H4K20 binding pocket on the surface of the Ankyrin repeats of TONSL, said method comprises use of at least part of the atomic co-ordinates data contained in PDB ID 53A4 or data derivable therefrom, wherein said method involves use of a computer modelling package or a computer program to model all or part of the structure of MCM2 HBD-G4-TONSL ARD in complex with H3 (57-135) and H4, thereby identifying said molecule by designing or selecting the molecule based on its likely ability to interact with a modelled structure.

20. An isolated polynucleotide or amino acid sequence having at least 90% sequence identity to any of SEQ ID NO 1-22.

21. A crystal comprising covalently linked MCM2 HBD—G₄—TONSL ARD in complex with H3 (57-135) and H4 (that diffracted to 2.43 Å resolution).

22. A crystal structure having the atomic coordinates or a subset hereof set out in PDB ID 5JA4 or having a structure in which the atomic coordinates vary by less than 3Å in any direction from those set out therein. 

1. An inhibitor of TONSL, wherein said inhibitor inhibits binding of TONSL ARD to histone H4. 2.-3. (canceled)
 4. The inhibitor according to claim 1, wherein the inhibitor is a small molecule, which targets the conformational space of the TONSL ARD occupied by the histone H4 tail encompassing residues K12-R23 and acting to prevent or disrupt the binding of the histone H4 tail K12-R23 with the TONSL ARD via direct competition or via allosteric disruption of the binding pocket. 5.-10. (canceled)
 11. The inhibitor according to claim 1, wherein the inhibitor is a peptide or a polypeptide optionally linked to a conjugated moiety.
 12. The inhibitor according to claim 11, wherein the inhibitor is a peptide comprising the sequence Arg-His-Xaa-Lys, wherein Xaa may be any amino acid.
 13. (canceled)
 14. The inhibitor according to claim 11, wherein the inhibitor is a peptide comprising the sequence Val-Leu-Arg. 15.-23. (canceled)
 24. The inhibitor according to claim 11, wherein the amino acid corresponding to Lys20 of SEQ ID NO:23 is unmethylated.
 25. The inhibitor according to claim 11, wherein said peptide comprises or consists of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22,
 26. The inhibitor according to claim 11, wherein said peptide comprises or consists of amino acids 512 to 692 of SEQ ID NO:16.
 27. The inhibitor according to claim 11, wherein the C-terminal of the peptide is amidated or aikylated and/or the N-terminal of the peptide is acetylated or formylated.
 28. (canceled)
 29. The inhibitor according to claim 11, wherein said conjugated moiety is a polymeric molecule, such as polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP).
 30. The inhibitor according to claim 1, wherein the inhibitor is a compound having a 3-[(3-Aminocyclopentyl)carbonyl]-1H-quinolin-4-one core.
 31. The inhibitor according to claim 1, wherein the TONSL ARD is identified as amino acids 512-692 of TONSL of SEQ ID NO:16.
 32. The inhibitor according to claim 1, wherein histone H4 is is histone H4 of SEQ ID NO:23 or histone H4 of SEQ ID NO:34.
 33. The inhibitor according to claim 4, wherein the K12-R23 of the histone H4 tail corresponds to amino acids 12 to 23 of SE Q ID NO:34.
 34. A method of treatment of a clinical condition, said method comprising admintstering a therapeutically effective amount of the inhibitor according to claim 1 to an individual in need thereof.
 35. A method of treatment of cancer, said method comprising administering a therapeutically effective amount of the inhibitor according to claim to an individual in need thereof. 36.-46. (canceled)
 47. An isolated polynucleotide or amino acid sequence having at least 90% sequence identity to any of SEQ ID NO 1-22.
 48. A TONSL mutant polypeptide comprising at least one mutation in an amino acid of the histone H4 tail binding surface of the TONSL ARD, wherein said TONSL mutant polypeptide apart from said mutation is identical to SEQ ID NO:16 or shares a sequence identity with SEQ ID NO:16 of at least 70%, wherein said TONSL mutant polypeptide is capable of binding MMS22L and wherein said TONSL mutant does not bind histone H4.
 49. The polypeptide according to claim 48 wherein said TONSL mutant polypeptide carries a mutation in one or more of the amino acids selected from the group consisting of: Glu530, Asp559, Trp563, Glu568, Gln571 and D604,
 50. The polypeptide according to claim 48, wherein the polypeptide comprises or consists of a sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO 21 and SEQ NO:22, 51.-63. (canceled) 